Oxopiperazine helix mimetics for control of Hypoxia-Inducible gene expression

ABSTRACT

The present invention relates to oxopiperazines that mimic helix αB of the C-terminal transactivation domain of HIF1α. Also disclosed are pharmaceutical compositions containing these oxopiperazines and methods of using these oxopiperazines (e.g., to reduce gene transcription, treat or prevent disorders mediated by interaction of HIF1a with CREB-binding protein and/or p300, reduce or prevent angiogenesis in a tissue, induce apoptosis, and decrease cell survival and/or proliferation).

This application is a national stage application under 35 U.S.C. § 371of International Application No. PCT/US2015/031816, filed, May 20, 2015,which claims the benefit of U.S. Provisional Patent Application Ser. No.62/001,530, filed May 21, 2014, which is hereby incorporated byreference in its entirety.

This invention was made with government support under CHE-1151554awarded by National Science Foundation and RC4-AI092765, PN2-EY016586,IU54CA143907-01, and EY016586-06 awarded by National Institutes ofHealth. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to oxopiperazines that substantially mimichelix αB of the C-terminal transactivation domain of Hypoxia-InducibleFactor 1α.

BACKGROUND OF THE INVENTION

Protein-protein interactions are often mediated by amino acid residuesorganized on secondary structures (Jones & Thornton, Prog. Biophys. Mol.Bio. 63:31 (1995)). Designed oligomeric ligands that can mimic the arrayof protein-like functionality at interfaces offer an attractive approachto target therapeutically important interactions (Ko et al., Chem. Soc.Rev. 40:4411 (2011)). Efforts to mimic interfacial α-helices haveresulted in three overarching synthetic strategies: helix stabilization,helical foldamers, and helical surface mimetics (Azzarito et al., Nat.Chem. 5:161 (2013); Henchey et al., Curr. Opin. Chem. Biol. 12:692(2008)).

Helix stabilization employs side chain crosslinks (Schafmeister et al.,J. Am. Chem. Soc. 122:5891 (2000); Harrison et al., Proc. Nat'l Acad.Sci. U.S.A. 107:11686 (2010)) or hydrogen-bond surrogates (Patgiri etal., Acc. Chem. Res. 41:1289 (2008)) to preorganize amino acid residuesand initiate helix formation. Helical foldamers are nonnatural oligomersthat adopt defined helical conformations (Gellman, Acc. Chem. Res.31:173 (1998); Goodman et al., Nat. Chem. Biol. 3:252 (2007)). Prominentexamples include β-peptide (Cheng et al., Chem. Rev. 101:3219 (2001);Horne & Gellman, Acc. Chem. Res. 41:1399 (2008); Seebach & Gardiner,Acc. Chem. Res. 41:1366 (2008)) and peptoid helices (Yoo & Kirshenbaum,Curr. Opin. Chem. Biol. 12:714 (2008)). Helical surface mimetics utilizeconformationally restricted scaffolds with attached functional groupsthat mimic the topography of α-helical side chains. With the exceptionof some elegant examples (Marimganti et al., Org. Lett. 11:4418 (2009);Jayatunga et al., Biorg. Med. Chem. Lett. 24:717 (2014); Thompson &Hamilton, Org. Biomol. Chem. 10:5780 (2012); Thompson et al.,Tetrahedron 68:4501 (2012); Jung et al., Org. Lett. 15:3234 (2013)),surface mimetics typically impart functionality from one face of thehelix (Marimganti et al., Org. Lett. 11:4418 (2009)), while stabilizedpeptide helices and foldamers are able to reproduce functionalitypresent on multiple faces of the target helix. A key advantage of helixsurface mimicry is that it affords low molecular weight compounds asmodulators of protein interactions (Plante et al., Chem. Commun. 5091(2009); Shaginian et al., J. Am. Chem. Soc. 131:5564 (2009); Restorp &Rebek, Bioorg. Med. Chem. Lett. 18:5909 (2008); Tošovská & Arora, Org.Lett. 12:1588 (2010); Buhrlage et al., ACS Chem. Biol. 4:335 (2009); Leeet al., J. Am. Chem. Soc. 133:676 (2011)).

A recent survey of protein-protein complexes in the Protein Data Bank(PDB) suggests that a significant portion of interface helices use oneface to target the binding partner (Bullock et al., J. Am. Chem. Soc.133:14220 (2011); Jochim & Arora, ACS Chem. Biol. 5:919 (2010)). Thisanalysis points to the meaningful role that topographical helix mimicscan play in affording small molecule inhibitors of protein-proteininteractions. The classical examples of helix surface mimics weredescribed by Hamilton et al. (Cummings & Hamilton, Curr. Opin. Chem.Biol. 14:341 (2010); Yin & Hamilton, Angew. Chem. Int'l Ed. 44:4130(2005); Orner et al., J. Am. Chem. Soc. 123:5382 (2001), and containedaromatic scaffolds displaying protein-like functionality (Azzarito etal., J. Nat. Chem. 5:161 (2013)). Oxopiperazines have since beenproposed as a new class of helix mimetics (FIGS. 1A-B) (Tošovská &Arora, Org. Lett. 12:1588 (2010)). Oxopiperazine-based scaffolds offerchiral backbones and can be easily assembled from α-amino acids allowingrapid diversification of the scaffold. 2-oxopiperazines anddiketopiperazines have a rich history in medicinal chemistry (Tošovská &Arora, Org. Lett. 12:1588 (2010); Gante, Angew. Chem. Intl Ed. Engl.33:1699 (1994); Giannis & Kolter, Angew. Chem. Int'l Ed. 32:1244 (1993);Herrero et al., J. Org. Chem. 67:3866 (2002); Kitamura et al., J. Med.Chem. 44:2438 (2001); Sugihara et al., J. Med. Chem. 41:489 (1998);Borthwick et al., J. Med. Chem. 49:4159 (2006).

As illustrated in FIG. 2, angiogenesis, the induction of new bloodvessels, is critical for normal growth as well as pathogenesis ofvarious disorders. In cancers, angiogenesis accelerates growth of solidtumors and provides a gateway to metastasis via the newly formedvasculature. In contrast, therapeutic angiogenesis is important forreducing the effects of tissue ischemia and preventing organ failure.

The process of angiogenesis is tightly controlled by a number ofspecific mitogens, among which vascular endothelial growth factor (VEGF)and its receptors play a key role. The levels of VEGF are upregulatedacross a broad range of tumors, and play a causal role in oncogenicsignaling. In cells and tissues, transcription of VEGF gene is regulatedby hypoxia-inducible factors. Among them, Hypoxia-Inducible Factor 1(“HIF1”) is the main regulator of oxygen-dependent transcription in amajority of organs and accounts for the increase in expression ofhypoxia-inducible genes.

HIF1 consists of an oxygen-sensitive a and a constitutively expressed βsubunit. Under well-oxygenated conditions, HIF1α is hydroxylated (Ivanet al., Science 292:464-8 (2001)), ubiquitinated, and degraded by theubiquitin-proteasome system. Under hypoxia, HIF1α is stabilized andtranslocates into the nucleus where heterodimerization with itsconstitutively expressed binding partner, aryl hydrocarbon receptornuclear translocator (“ARNT”) (Wood et al., J. Biol. Chem. 271:15117-23(1996)), results in binding to a cognate hypoxia response element(“HRE”) (Forsythe et al., Mol. Cell. Biol. 16:4604-13 (1996)). Theheterodimer then recruits transcriptional coactivators, p300, CBP, andSRC-1, resulting in the upregulation of the hypoxia-inducible genes.

Regulation of the activity of hypoxia-inducible factors includes threecritical steps: (i) inhibition of hydroxylation of two proline residuesto preclude interaction of HIF1α with pVHL, a part of ubiquitin ligasecomplex, thereby preventing its proteasomal destruction; (ii) inhibitionof hydroxylation of Asn803 by Factor Inhibiting HIF1α (“FIH”) (Lando etal., Genes Develop. 16:1466-71 (2002)) to enable recruitment ofcoactivators, which trigger overexpression of hypoxia inducible genes,including genes encoding angiogenic peptides such as VEGF and VEGFreceptors VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), as well as proteinsinvolved in altered energy metabolism, such as the glucose transportersGLUT1 and GLUT3, and hexokinases 1 and 2 (Forsythe et al., Mol. Cell.Biol. 16:4604-13 (1996); Okino et al., J. Biol. Chem. 273:23837-43(1998)); and (iii) interaction of promoter-bound HIF1α with coactivatorprotein p300 (or the homologous CREB binding protein, CBP) leading toupregulation of transcription.

The interaction between the cysteine-histidine rich 1 domain (“CH1”) ofp300/CBP and the C-terminal transactivation domain (“C-TAD₇₈₆₋₈₂₆”) ofHIF1α (Freedman et al., Proc. Nat'l Acad. Sci. USA 99:5367-72 (2002);Dames et al., Proc. Nat'l Acad. Sci. USA 99:5271-6 (2002)) mediatestransactivation of hypoxia-inducible genes (Hirota & Semenza, Crit. Rev.Oncol. Hematol. 59:15-26 (2006); Semenza, Nat. Rev. Cancer 3:721-32(2003)) (see FIG. 3A). As illustrated in FIGS. 3A-C, structural studiesprovide a molecular basis for this transcription factor-coactivatorinteraction and identify two short α-helical domains from HIF1α as keydeterminants for its recognition by p300 (Freedman et al., Proc. Nat'lAcad. Sci. USA 99:5367-72 (2002); Dames et al., Proc. Nat'l Acad. Sci.U.S.A. 99:5271-76 (2002)). Synthetic mimics of these domains couldinhibit HIF1α/p300 or HIF1α/CBP complex formation and regulatetranscription. Key residues contributing to the binding of one of thetwo helices (PDB code 1L8C, residues 139-147) are shown in FIG. 3C.

Because interaction of HIF1α C-TAD with transcriptional coactivatorp300/CBP is a point of significant amplification in transcriptionalresponse, its disruption with designed ligands could be an effectivemeans of suppressing aerobic glycolysis and angiogenesis (i.e., theformation of new blood vessels) in cancers (Hirota & Semenza, Crit. Rev.Oncol. Hematol. 59:15-26 (2006); Rarnanathan et al., Proc. Nat'l Acad.Sci. USA 102:5992-7 (2005); Underiner et al., Curr. Med. Chem. 11:731-45(2004)).

The present invention is directed to overcoming these and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an oxopiperazine selectedfrom the group consisting of

-   (i) Formula IA:

-   wherein:-   R₁ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₁ and R₂    are not both an aryl;-   R₃ is hydrophobic and is an amino acid side chain, SR, or an alkyl;    wherein R is independently H, an alkyl, or an aryl;-   R₄ is a hydrogen bond donor, a hydrophobic amino acid side chain, or    an amide;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   X₁ is H, N(R)₂, OR, COR′, CO₂R′, CONR′, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a peptide of 1 to about 10    amino acid residues, a protecting group for protection of an amine,    a solubilizing group, a targeting moiety, or a tag; wherein each R    is independently H, an alkyl, or an aryl; and wherein R′ is H, an    alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a    targeting moiety, or a tag; with the proviso that X₁ is absent when    Z is O or S;-   Z is N, O, or S;-   A₁-W₁ is:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to about 10 amino acid    residues, a protecting group for protection of a carboxylic acid, a    targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;    and wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag;-   (ii) Formula IB:

-   wherein:-   R₀ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₁ and R₂ are each independently a solubilizing group, a hydrophobic    amino acid side chain, H, N(R)₂, OR, SR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   R₃ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₀ and R₃    are not both an aryl;-   R₄ is a hydrogen bond donor, a hydrophobic amino acid side chain, or    an amide;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   X′ is H, COR′, CO₂R′, CONR′, OR′, N(R″)₂, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a solubilizing group, a    targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, an amino acid residue, a    peptide of 1 to about 10 amino acid residues, a targeting moiety, or    a tag; and wherein each R″ is independently H, CO₂R′, CONR′, an    alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a    targeting moiety, or a tag;-   A₁-W₁ is:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to about 10 amino acid    residues, a protecting group for protection of a carboxylic acid, a    targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;    and wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag;-   and (iii) Formula IC:

-   wherein:-   R₁ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₁ and R₂    are not both an aryl;-   R₃ is hydrophobic and is an amino acid side chain, SR, or an alkyl;    wherein R is independently H, an alkyl, or an aryl;-   R₄ is a hydrogen bond donor or an amide;-   R₅ is a hydrophobic amino acid side chain;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   R₇ is a solubilizing group, a hydrophobic amino acid side chain, H,    N(R)₂, OR, SR, halogen, an alkyl, or an aryl; wherein each R is    independently H, an alkyl, or an aryl;-   X₁ is H, N(R)₂, OR, COR′, CO₂R′, CONR′, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a peptide of 1 to about 10    amino acid residues, a protecting group for protection of an amine,    a solubilizing group, a targeting moiety, or a tag; wherein each R    is independently H, an alkyl, or an aryl; and wherein R′ is H, an    alkyl, an aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a    targeting moiety, or a tag; with the proviso that X₁ is absent when    Z is O or S;-   Z is N, O or S;-   each A₁-W₁ is independently:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to about 10 amino acid    residues, a protecting group for protection of a carboxylic acid, a    targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;    and wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag.

The present invention is further directed to pharmaceutical formulationscontaining the oxopiperazine of Formula IA, Formula IB, or Formula IC.

A second aspect of the present invention relates to a method of reducingtranscription of a gene in a cell, where transcription of the gene ismediated by interaction of HIF1α with CREB-binding protein and/or p300.This method involves contacting the cell with an oxopiperazine of thepresent invention under conditions effective to reduce transcription ofthe gene.

A third aspect of the present invention relates to a method of treatingor preventing in a subject a disorder mediated by interaction of HIF1αwith CREB-binding protein and/or p300. This method involvesadministering an oxopiperazine of the present invention to the subjectunder conditions effective to treat or prevent the disorder.

A fourth aspect of the present invention relates to a method of reducingor preventing angiogenesis in a tissue. This method involves contactingthe tissue with an oxopiperazine of the present invention underconditions effective to reduce or prevent angiogenesis in the tissue.

A fifth aspect of the present invention relates to a method of inducingapoptosis of a cell. This method involves contacting the cell with anoxopiperazine of the present invention under conditions effective toinduce apoptosis of the cell.

A sixth aspect of the present invention relates to a method ofdecreasing survival and/or proliferation of a cell. This method involvescontacting the cell with an oxopiperazine of the present invention underconditions effective to decrease survival and/or proliferation of thecell.

The potential of oxopiperazine helix mimetics (OHMs) to targetprotein-protein interactions was recently established in biochemical,cell culture, and in vivo assays (Lau et al., Proc. Nat'l Acad. Sci.10.1073/pnas.1402393111 (published online May 14, 2014); InternationalPatent Application No. PCT/US13/26722 to Arora et al., each of which ishereby incorporated by reference in its entirety). It was shown thatOHMs that mimic a key α-helix from HIF1α can inhibit the interactions ofthis transcription factor with coactivator p300/CBP. Significantly, thedesigned compounds downregulate the expression of a specific set ofgenes and reduce tumor burden in mouse xenograft models. Encouraged bythis success, it was sought to develop a computational approach todesign and optimize oxopiperazine analogs with natural and nonnaturalamino acid residues.

The objective of computational molecular design is to reduce the totalnumber of possible designs to a manageable number that can beefficiently synthesized and experimentally tested. Contemporarycomputational methods for design of protein-protein interactioninhibitors often emphasize fragment-based screening (Schneider &Fechner, Nat. Rev. Drug Discov. 4:649 (2005); Winter et al., Quart. Rev.Biophys. 45:383 (2012), each of which is hereby incorporated byreference in its entirety). As a complementary approach, peptidomimeticdesign seeks to graft appropriate side chains on stable syntheticbackbones, i.e., helical or β-sheet scaffolds.

The new computational protocol was used to develop nanomolar ligands fortwo different protein-protein interactions. Described herein is thedesign of oxopiperazine dimers that mimic HIF1α to develop ligands forp300/CBP. The previous design of oxopiperazine analogs of HIF1α wasbased on mimicry of natural residues and resulted in sub-micromolarinhibitors. As described herein, application of the new Rosetta basedpeptidomimetic design strategy with noncanonical residues affordsoxopiperazine helix mimetics that are greater than an order of magnitudemore potent than the scaffolds that displayed wild-type residues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-B relate to the design of oxopiperazine helix mimetics. FIG. 1Aillustrates the design of amino acid-derived oxopiperazines (Tošovskaá&Arora, Org. Lett. 12:1588 (2010), which is hereby incorporated byreference in its entirety). The oxopiperazines are obtained by linkingneighboring amide nitrogen atoms in peptides with ethylene bridges, asdepicted. FIG. 1B is an overlay of an 8-mer canonical α-helix and anoxopiperazine dimer (left), and a predicted low energy structure of anoxopiperazine dimer (right). Side chain groups are depicted as spheres.

FIG. 2 is a schematic illustration of HIF1α-mediated regulation ofoxygen-dependent transcription (Rankin & Giaccia, Cell Death Different.15:678 (2008), which is hereby incorporated by reference in itsentirety).

FIGS. 3A-C relate to the HIF1α/TAZ1 structure (Dames et al. Proc. Nat'lAcad. Sci. 99:5271 (2002), which is hereby incorporated by reference inits entirety). FIG. 3A is a schematic diagram illustrating the structureof the complex of the C-terminal transactivation domain (“C-TAD”) ofHIF1α with cysteine-histidine rich 1 domain (“CH1”) of the coactivatorprotein p300. The human HIF1α C-TAD sequence (SEQ ID NO: 1) is shown inFIG. 3B, along with the location of the αA helix (PDB residues 121-127)and αB helix (PDB residues 139-147). FIG. 3C is a table showing the keyresidues contributing to the binding of helix αB.

FIG. 4 is a schematic diagram illustrating the interaction betweentranscription factors, coactivators, and promoters in initiatingtranscription (Guarracino et al., Biopolymers 95:1 (2011), which ishereby incorporated by reference in its entirety).

FIG. 5 is a violin plot showing distribution of the predictedoxopiperazine analogs for their potential to target the CH1 domain ofp300/CBP. The binding affinity is expressed as Rosetta binding energyunit (REU). The plot shows the energy scores for the top scoring 1,000designs selected from 30,000 random Rosetta designs (gray violin) aswell as experimentally tested designs (dots). The Rosetta scorediscriminates between good binders (green and yellow label) and weakbinders (red label).

FIGS. 6A-E are analytical HPLC traces of the indicated oxopiperazinesand monomer-peptides. For compound 21, HPLC was performed in 5% to 95%acetonitrile in water (0.1% formic acid) for 30 minutes. The UV trace isat 220 nm. For compound 22, HPLC was performed in 5% to 95% acetonitrilein water (0.1% trifluoroacetic acid) for 10 minutes; 95% to 100% from10-15 minutes. The UV trace is at 230 nm. For compounds 23-25, HPLC wasperformed in 5% to 95% acetonitrile in water (0.1% trifluoroacetic acid)for 10 minutes. The UV traces are at 220 nm.

FIG. 7 relates to the design of HIF1α mimetics as ligands for p300-CH1.FIG. 7 is an overlay of HIF1α helix₇₇₆₋₈₂₆ (in magenta) and OHM 21 incomplex with the CH1 domain of p300/CBP (PDB code 1L8C). The R₁, R₂ andR₄ positions of OHM 21 access the same p300 molecular pockets as Leu818,Leu822, and Gln824 of the HIF1α C-terminal activation domain.

FIG. 8 is a graph of the binding affinity for p300-CH1 determined bytryptophan fluorescence spectroscopy. Binding curves for compounds OHM21-25 are shown.

FIGS. 9A-D are space-filling models of the R₂ position of HIF OHM mimicsin the p300-CH1 binding pocket, showing leucine of OHM 21 (FIG. 9A),alanine of OHM 22 (FIG. 9B), homoleucine of OHM 25 (FIG. 9C), andnorleucine of OHM 23 (FIG. 9D). The space-filling model reveals thatlonger hydrophobic side chains form better packing in the p300-CH1pocket, natively inhabited by the Leu822 of HIF1α.

FIG. 10 is a graph showing the correlation of Rosetta binding energypredictions with experimental K_(d) for p300-CH1 ligands. Data pointsare taken from Table 8, and the correlation coefficient was calculatedusing Python's scipy.stats.stats.pearsonr function. Rosetta bindingenergy (x axis) approximates free energy of binding which isproportional to ln(Kd). Explicitly: deltaG=RTln(Kd) where R=ideal gasconstant and T=temperature.

FIGS. 11A-B are graphs of oxopiperazine analog binding to His₆-taggedMdm2 determined by a fluorescence-polarization assay (FIG. 11A) and top300-CH1 determined by tryptophan fluorescence spectroscopy (FIG. 11B).

FIG. 12 shows the structure of p53 mimic OHM 18.

FIG. 13 is a violin plot showing the distribution of the predictedoxopiperazine analogs for their potential to target Mdm2. The bindingaffinity is expressed as Rosetta binding energy unit (REU). The plotshows the energy scores for the top scoring 1,000 designs selected from30,000 random Rosetta designs (gray violin) as well as experimentallytested designs (dots). The Rosetta score discriminates between goodbinders (green and yellow label) and weak binders (red label).

DETAILED DESCRIPTION OF THE INVENTION

As illustrated in FIG. 4, transcription factors are involved in anintricate web of interactions with partner proteins and promoter DNA,which result in recruitment of chromatin-remodeling enzymes and assemblyof the preinitiation complex (MARK PTASHNE & ALEXANDER GANN, GENES ANDSIGNALS (2002), which is hereby incorporated by reference in itsentirety). Because of the essential role gene expression plays in theprogression of diseases, synthetic agents that modulate transcription ina defined manner are attractive candidates for drug design (Arndt, AngewChem. Int'l Ed. Engl. 45:4552-60 (2006); Berg, “Curr. Opin. Chem. Biol.12:464-71 (2008); Mapp, Org. Biomol. Chem. 1:2217-20 (2003), each ofwhich is hereby incorporated by reference in its entirety). Describedherein is the design of oxopiperazine helix mimetics (OHMs) that inhibittranscription of hypoxia inducible genes by modulating the interactionbetween HIF1α transcription factor and coactivator p300/CBP. Theseoxopiperazines mimic helix αB of the C-terminal transactivation domain(“C-TAD”) of HIF1α and include one or more side chains from anon-natural amino acid.

One aspect of the present invention relates to an oxopiperazine selectedfrom the group consisting of

-   (i) Formula IA:

-   wherein:-   R₁ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₁ and R₂    are not both an aryl;-   R₃ is hydrophobic and is an amino acid side chain, SR, or an alkyl;    wherein R is independently H, an alkyl, or an aryl;-   R₄ is a hydrogen bond donor, a hydrophobic amino acid side chain, or    an amide;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   X₁ is H, N(R)₂, OR, COR′, CO₂R′, CONR′, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a peptide of 1 to 5 amino    acid residues, a peptide of 1 to 6 amino acid residues, a peptide of    1 to 7 amino acid residues, a peptide of 1 to 8 amino acid residues,    a peptide of 1 to 9 amino acid residues, a peptide of 1 to 10 amino    acid residues, a peptide of 1 to about 10 amino acid residues, a    protecting group for protection of an amine, a solubilizing group, a    targeting moiety, or a tag; wherein each R is independently H, an    alkyl, or an aryl; and wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;    with the proviso that X₁ is absent when Z is O or S;-   Z is N, O, or S;-   A₁-W₁ is:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to 5 amino acid residues,    a peptide of 1 to 6 amino acid residues, a peptide of 1 to 7 amino    acid residues, a peptide of 1 to 8 amino acid residues, a peptide of    1 to 9 amino acid residues, a peptide of 1 to 10 amino acid    residues, a peptide of 1 to about 10 amino acid residues, a    protecting group for protection of a carboxylic acid, a targeting    moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl,    a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and    wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag;-   (ii) Formula IB:

-   wherein:-   R₀ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₁ and R₂ are each independently a solubilizing group, a hydrophobic    amino acid side chain, H, N(R)₂, OR, SR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   R₃ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₀ and R₃    are not both an aryl;-   R₄ is a hydrogen bond donor, a hydrophobic amino acid side chain, or    an amide;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   X′ is H, COR′, CO₂R′, CONR′, OR′, N(R″)₂, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a solubilizing group, a    targeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, an amino acid residue, a    peptide of 1 to 5 amino acid residues, a peptide of 1 to 6 amino    acid residues, a peptide of 1 to 7 amino acid residues, a peptide of    1 to 8 amino acid residues, a peptide of 1 to 9 amino acid residues,    a peptide of 1 to 10 amino acid residues, a peptide of 1 to about 10    amino acid residues, a targeting moiety, or a tag; and wherein each    R″ is independently H, CO₂R′, CONR′, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;-   A₁-W₁ is:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to 5 amino acid residues,    a peptide of 1 to 6 amino acid residues, a peptide of 1 to 7 amino    acid residues, a peptide of 1 to 8 amino acid residues, a peptide of    1 to 9 amino acid residues, a peptide of 1 to 10 amino acid    residues, a peptide of 1 to about 10 amino acid residues, a    protecting group for protection of a carboxylic acid, a targeting    moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl,    a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and    wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag;-   and (iii) Formula IC:

-   wherein:-   R₁ is hydrophobic and is an amino acid side chain, OR, SR, an alkyl,    or an aryl; wherein R is independently H, an alkyl, or an aryl;-   R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl;    wherein R is independently H, an alkyl, or an aryl; wherein R₂ is    either a leucine side chain or longer than a leucine side chain by    at least one backbone methylene; and with the proviso that R₁ and R₂    are not both an aryl;-   R₃ is hydrophobic and is an amino acid side chain, SR, or an alkyl;    wherein R is independently H, an alkyl, or an aryl;-   R₄ is a hydrogen bond donor or an amide;-   R₅ is a hydrophobic amino acid side chain;-   each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an    aryl; wherein each R is independently H, an alkyl, or an aryl;-   R₇ is a solubilizing group, a hydrophobic amino acid side chain, H,    N(R)₂, OR, SR, halogen, an alkyl, or an aryl; wherein each R is    independently H, an alkyl, or an aryl;-   X₁ is H, N(R)₂, OR, COR′, CO₂R′, CONR′, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a peptide of 1 to 5 amino    acid residues, a peptide of 1 to 6 amino acid residues, a peptide of    1 to 7 amino acid residues, a peptide of 1 to 8 amino acid residues,    a peptide of 1 to 9 amino acid residues, a peptide of 1 to 10 amino    acid residues, a peptide of 1 to about 10 amino acid residues, a    protecting group for protection of an amine, a solubilizing group, a    targeting moiety, or a tag; wherein each R is independently H, an    alkyl, or an aryl; and wherein R′ is H, an alkyl, an aryl, an    arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;    with the proviso that X₁ is absent when Z is O or S;-   Z is N, O, or S;-   each A₁-W₁ is independently:

and

-   Y is OR′, N(R″′)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, a    heteroaryl, an amino acid, a peptide of 1 to 5 amino acid residues,    a peptide of 1 to 6 amino acid residues, a peptide of 1 to 7 amino    acid residues, a peptide of 1 to 8 amino acid residues, a peptide of    1 to 9 amino acid residues, a peptide of 1 to 10 amino acid    residues, a peptide of 1 to about 10 amino acid residues, a    protecting group for protection of a carboxylic acid, a targeting    moiety, or a tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl,    a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and    wherein each R″′ is independently H, CO₂R′, CONR′, an alkyl, an    aryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety,    or a tag.

Amino acid side chains according to this and all aspects of the presentinvention can be any amino acid side chain—from natural or nonnaturalamino acids—including alpha amino acids, disubstituted amino acids, betaamino acids, gamma amino acids, L-amino acids, D-amino acids,halogenated amino acids, etc. Hydrophobic amino acid side chains arewell known in the art and include, for example, phenylalanine,tryptophan, leucine, alanine, isoleuceine, valine, tyrosine, norleucine,homoleucine, 3-chloro-phenylalanine, naphthaline,3-methyl-phenylalanine, 4-chloro-phenylalanine, and (O—R)-tyrosine.

As used herein, the term “alkyl” means an aliphatic hydrocarbon groupwhich may be straight or branched having about 1 to about 6 carbon atomsin the chain, unless otherwise specified. Branched means that one ormore lower alkyl groups such as methyl, ethyl or propyl are attached toa linear alkyl chain. Exemplary alkyl groups include methyl, ethyl,n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.

As used herein, the term “cycloalkyl” refers to a non-aromatic saturatedor unsaturated mono- or polycyclic ring system which may contain 3 to 6carbon atoms, and which may include at least one double bond. Exemplarycycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclohexenyl, anti-bicyclopropane, or syn-bicyclopropane.

As used herein, the term “aryl” refers to an aromatic monocyclic orpolycyclic ring system containing from 6 to 19 carbon atoms, where thering system may be optionally substituted. Aryl groups of the presentinvention include, but are not limited to, groups such as phenyl,naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl,triphenylenyl, chrysenyl, and naphthacenyl.

As used herein, the term “arylalkyl” refers to a radical of the formula—R^(a)R^(b) where R^(a) is an alkyl radical as defined above and R^(b)is an aryl radical as defined above. The alkyl radical and thecycloalkyl radical may be optionally substituted as defined above.

As used herein, “heteroaryl” refers to an aromatic ring radical whichconsists of carbon atoms and from one to five heteroatoms selected fromthe group consisting of nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups include, without limitation, pyrrolyl, pyrazolyl,imidazolyl, triazolyl, furyl, thiophenyl, oxazolyl, isoxazolyl,thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, triazinyl, thienopyrrolyl, furopyrrolyl,indolyl, azaindolyl, isoindolyl, indolinyl, indolizinyl, indazolyl,benzimidazolyl, imidazopyridinyl, benzotriazolyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, pyrazolopyridinyl, triazolopyridinyl,thienopyridinyl, benzothiadiazolyl, benzofuyl, benzothiophenyl,quinolinyl, isoquinolinyl, tetrahydroquinolyl, tetrahydroisoquinolyl,cinnolinyl, quinazolinyl, quinolizilinyl, phthalazinyl, benzotriazinyl,chromenyl, naphthyridinyl, acrydinyl, phenanzinyl, phenothiazinyl,phenoxazinyl, pteridinyl, and purinyl. Additional heteroaryls aredescribed in COMPREHENSIVE HETEROCYCLIC CHEMISTRY: THE STRUCTURE,REACTIONS, SYNTHESIS AND USE OF HETEROCYCLIC COMPOUNDS (Katritzky et al.eds., 1984), which is hereby incorporated by reference in its entirety.

Solubilizing groups according to this and all aspects of the presentinvention include, without limitation, lysine, arginine, andpoly(ethylene glycol).

The oxopiperazines of Formula IA, Formula IB, or Formula IC may comprisea protecting group that is suitable for the protection of an amine or acarboxylic acid. Such protecting groups function primarily to protect ormask the reactivity of functional groups. Protecting groups that aresuitable for the protection of an amine group are well known in the art,including without limitation, carbamates, amides, N-alkyl and N-arylamines, imine derivatives, enamine derivatives, and N-hetero atomderivatives as described by THEODORA W. GREENE & PETER G. M. WUTS,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 494-615 (1999), which is herebyincorporated by reference in its entirety. Protecting groups that aresuitable for the protection of a carboxylic acid are also well known inthe art. Suitable carboxylic acid protecting groups include, withoutlimitation, esters (e.g., substituted methyl esters, 2-substituted ethylesters, 2,6-dialkylphenyl esters, substituted benzyl esters, silylesters, and stannyl esters), amides, and hydrazides as described byTHEODORA W. GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANICSYNTHESIS 372-450 (1999), which is hereby incorporated by reference inits entirety. Methods of protecting and deprotecting amine andcarboxylic acids vary depending on the chosen protecting group; however,these methods are well known in the art and described in THEODORA W.GREENE & PETER G. M. WUTS, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS372-450 and 494-615 (1999), which is hereby incorporated by reference inits entirety.

A targeting moiety according to the present invention functions to (i)promote the cellular uptake of the oxopiperazine, (ii) target theoxopiperazine to a particular cell or tissue type (e.g., signalingpeptide sequence), or (iii) target the oxopiperazine to a specificsub-cellular localization after cellular uptake (e.g., transport peptidesequence).

To promote the cellular uptake of an oxopiperazine of the presentinvention, the targeting moiety may be a cell penetrating peptide (CPP).CPPs translocate across the plasma membrane of eukaryotic cells by aseemingly energy-independent pathway and have been used successfully forintracellular delivery of macromolecules, including antibodies,peptides, proteins, and nucleic acids, with molecular weights severaltimes greater than their own. Several commonly used CPPs, includingpolyarginines, transportant, protamine, maurocalcine, and M918, aresuitable targeting moieties for use in the present invention and arewell known in the art (see Stewart et al., Org. Biomol. Chem.6:2242-2255 (2008), which is hereby incorporated by reference in itsentirety). Additionally, methods of making CPP are described in U.S.Patent Application Publication No. 20080234183 to Hallbrink et al.,which is hereby incorporated by reference in its entirety.

Another suitable targeting moiety useful for enhancing the cellularuptake of the oxopiperazine is an “importation competent” signal peptideas disclosed by U.S. Pat. No. 6,043,339 to Lin et al., which is herebyincorporated by reference in its entirety. An importation competentsignal peptides is generally about 10 to about 50 amino acid residues inlength, typically hydrophobic residues, that render the oxopiperazinecapable of penetrating through the cell membrane from outside the cellto the interior of the cell. An exemplary importation competent signalpeptide includes the signal peptide from Kaposi fibroblast growth factor(see U.S. Pat. No. 6,043,339 to Lin et al., which is hereby incorporatedby reference in its entirety). Other suitable peptide sequences can beselected from the SIGPEP database (see von Heijne G., Protein Seq. DataAnal. 1(1):41-42 (1987), which is hereby incorporated by reference inits entirety).

Another suitable targeting moiety is a signal peptide sequence capableof targeting the oxopiperazine to a particular tissue or cell type. Thesignaling peptide can include at least a portion of a ligand bindingprotein. Suitable ligand binding proteins include high-affinity antibodyfragments (e.g., Fab, Fab′ and F(ab′)₂), single-chain Fv antibodyfragments), nanobodies or nanobody fragments, fluorobodies, or aptamers.Other ligand binding proteins include biotin-binding proteins,lipid-binding proteins, periplasmic binding proteins, lectins, serumalbumins, enzymes, phosphate and sulfate binding proteins,immunophilins, metallothionein, or various other receptor proteins. Forcell specific targeting, the signaling peptide is preferably a ligandbinding domain of a cell specific membrane receptor. Thus, when themodified oxopiperazine is delivered intravenously or otherwiseintroduced into blood or lymph, the oxopiperazine will adsorb to thetargeted cell, and the targeted cell will internalize the oxopiperazine.For example, if the target cell is a cancer cell, the oxopiperazine maybe conjugated to an anti-C3B(I) antibody as disclosed by U.S. Pat. No.6,572,856 to Taylor et al., which is hereby incorporated by reference inits entirety. Alternatively, the oxopiperazine may be conjugated to analphafeto protein receptor as disclosed by U.S. Pat. No. 6,514,685 toMoro, or to a monoclonal GAH antibody as disclosed by U.S. Pat. No.5,837,845 to Hosokawa, each of which is hereby incorporated by referencein its entirety. For targeting an oxopiperazine to a cardiac cell, theoxopiperazine may be conjugated to an antibody recognizing elastinmicrofibril interfacer (EMILIN2) (Van Hoof et al., J. Proteom. Res.9:1610-18 (2010), which is hereby incorporated by reference in itsentirety), cardiac troponin I, connexin-43, or any cardiac cell-surfacemembrane receptor that is known in the art. For targeting anoxopiperazine to a hepatic cell, the signaling peptide may include aligand domain specific to the hepatocyte-specific asialoglycoproteinreceptor. Such chimeric oxopiperazines can be prepared using similarmethods as those for preparing chimeric proteins and peptides describedin U.S. Pat. No. 5,817,789 to Heartlein et al., which is herebyincorporated by reference in its entirety.

Another suitable targeting moiety is a transport peptide that directsintracellular compartmentalization of the oxopiperazine once it isinternalized by a target cell or tissue. For example, if the proteinactivity or protein-protein interaction that is sought to be inhibitedoccurs in the endoplasmic reticulum (ER), the oxopiperazine can beconjugated to an ER transport peptide sequence. A number of such signalpeptides are known in the art, including the signal peptideMMSFVSLLLVGILFYATEAEQLTKCEVFQ (SEQ ID NO: 2). Other suitable ER signalpeptides include the N-terminus endoplasmic reticulum targeting sequenceof the enzyme 17β-hydroxysteroid dehydrogenase type 11 (Horiguchi etal., Arch. Biochem. Biophys. 479(2):121-30 (2008), which is herebyincorporated by reference in its entirety), or any of the ER signalingpeptides (including the nucleic acid sequences encoding the ER signalpeptides) disclosed in U.S. Patent Publication No. 20080250515 to Reedet al., which is hereby incorporated by reference in its entirety.Additionally, the oxopiperazine of the present invention can contain anER retention signal, such as the retention signal KEDL (SEQ ID NO: 3).Methods of modifying the oxopiperazines of the present invention toincorporate transport peptides for localization of the oligomers to theER can be carried out as described in U.S. Patent Publication No.20080250515 to Reed et al., which is hereby incorporated by reference inits entirety.

If the protein activity or protein-protein interaction that is sought tobe inhibited occurs in the nucleus, the oxopiperazine can include anuclear localization transport signal. Suitable nuclear transportpeptide sequences are known in the art, including the nuclear transportpeptide PPKKKRKV (SEQ ID NO: 4). Other nuclear localization transportsignals include, for example, the nuclear localization sequence ofacidic fibroblast growth factor and the nuclear localization sequence ofthe transcription factor NF-KB p50 as disclosed by U.S. Pat. No.6,043,339 to Lin et al., which is hereby incorporated by reference inits entirety. Other nuclear localization peptide sequences known in theart are also suitable for use in the accordance with this aspect of theinvention.

Suitable transport peptide sequences for targeting to the mitochondriainclude MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO: 5). Other suitabletransport peptide sequences suitable for selectively targeting theoxopiperazine of the present invention to the mitochondria are disclosedin U.S. Published Patent Application No. 20070161544 to Wipf, which ishereby incorporated by reference in its entirety.

A “tag” as used herein includes any labeling moiety that facilitates thedetection, quantitation, separation, and/or purification of theoxopiperazine of the present invention. Suitable tags includepurification tags, radioactive or fluorescent labels, and enzymatictags.

Purification tags, such as poly-histidine (His₆), aglutathione-S-transferase (GST-), or maltose-binding protein (MBP-), canassist in oligomer purification or separation but can later be removed,i.e., cleaved from the oxopiperazine following recovery.Protease-specific cleavage sites can be used to facilitate the removalof the purification tag. The desired oxopiperazine product can bepurified further to remove the cleaved purification tags.

Other suitable tags include radioactive labels, such as, ¹²⁵I, ¹³¹I,¹¹¹In, or ⁹⁹TC. Methods of radiolabeling compounds, are known in the artand described in U.S. Pat. No. 5,830,431 to Srinivasan et al., which ishereby incorporated by reference in its entirety. Radioactivity isdetected and quantified using a scintillation counter orautoradiography. Alternatively, the oxopiperazine can be conjugated to afluorescent tag. Suitable fluorescent tags include, without limitation,chelates (europium chelates), fluorescein and its derivatives, rhodamineand its derivatives, dansyl, Lissamine, phycoerythrin and Texas Red. Thefluorescent labels can be conjugated to the oxopiperazine usingtechniques disclosed in CURRENT PROTOCOLS IN IMMUNOLOGY (Coligen et al.eds., 1991), which is hereby incorporated by reference in its entirety.Fluorescence can be detected and quantified using a fluorometer.

Enzymatic tags generally catalyze a chemical alteration of a chromogenicsubstrate which can be measured using various techniques. For example,the enzyme may catalyze a color change in a substrate, which can bemeasured spectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Examples of suitableenzymatic tags include luciferases (e.g., firefly luciferase andbacterial luciferase; see e.g., U.S. Pat. No. 4,737,456 to Weng et al.,which is hereby incorporated by reference in its entirety), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases(e.g., horseradish peroxidase), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclicoxidases (e.g., uricase and xanthine oxidase), lactoperoxidase,microperoxidase, and the like. Techniques for conjugating enzymes toproteins and peptides are described in O'Sullivan et al., Methods forthe Preparation of Enzyme—Antibody Conjugates for Use in EnzymeImmunoassay, in METHODS IN ENZYMOLOGY 147-66 (Langone et al. eds.,1981), which is hereby incorporated by reference in its entirety, can beused for conjugating enzymes to oxopiperazines of the present invention.Such tags may be particularly useful for detecting inhibition ofprotein-protein interactions using the oxopiperazine of the presentinvention.

The oxopiperazines of Formula IA, Formula IB, or Formula IC can alsocomprise a hydrogen bond donor. Hydrogen bond donors contain anelectronegative atom with at least one proton to share. Suitableexamples include, without limitation, amine, amide, carboxylic acids,hydroxyl, and thiol functional groups.

In one embodiment of the present invention, the oxopiperazine has aformula of Formula IA. In a preferred embodiment, R₁ is OR, SR, a C₃-C₆alkyl, an aryl, or a side chain of an amino acid selected from the groupconsisting of leucine, methionine, and homoleucine. In a preferredembodiment, R₂ is OR, SR, a C₃-C₆ alkyl, an aryl, or a side chain of anamino acid selected from the group consisting of norleucine, methionine,leucine, and homoleucine. In a preferred embodiment, R₃ is SR, a C₁-C₃alkyl, or a side chain of an amino acid selected from the groupconsisting of glycine and alanine. In a preferred embodiment, R₄ is aside chain of an amino acid selected from the group consisting ofglutamine, alanine, valine, asparagine, serine, and homoserine. In apreferred embodiment, Y is OH, OR′, NHR′, NR′₂, or NH₂. Combinations ofthese embodiments are also contemplated. In further preferredembodiments, R₁, R₂, R₃, and R₄ are amino acid side chains as shown inTable 1 below. Combinations of these embodiments are also contemplated.

TABLE 1 Exemplary Embodiments of Formula IA Emb. R₁ R₂ R₃ R₄ A Leu NleAla Gln B Met Met Ala Gln C Hle Hle Ala Gln D Hle Leu Ala Gln E Met LeuAla Gln F Leu Hle Ala Gln G Hle Nle Ala Gln

In one embodiment of the present invention, the oxopiperazine has aformula of Formula IB. In a preferred embodiment, R₀ is OR, SR, a C₃-C₆alkyl, an aryl, or a side chain of an amino acid selected from the groupconsisting of leucine, methionine, and homoleucine. In a preferredembodiment, R₃ is OR, SR, a C₃-C₆ alkyl, an aryl, or a side chain of anamino acid selected from the group consisting of norleucine, methionine,leucine, and homoleucine. In a preferred embodiment, R₄ is a side chainof an amino acid selected from the group consisting of glutamine,alanine, valine, asparagine, serine, and homoserine. In a preferredembodiment, Y is OH, OR′, NHR′, NR′₂, or NH₂. Combinations of theseembodiments are also contemplated. In further preferred embodiments, R₀,R₃, and R₄ are amino acid side chains as shown in Table 2 below.

TABLE 2 Exemplary Embodiments of Formula IB Emb. R₀ R₃ R₄ A Leu Nle GlnB Met Met Gln C Hle Hle Gln D Hle Leu Gln E Met Leu Gln F Leu Hle Gln GHle Nle Gln

In one embodiment of the present invention, the oxopiperazine has aformula of Formula IC. In a preferred embodiment, R₁ is OR, SR, a C₃-C₆alkyl, an aryl, or a side chain of an amino acid selected from the groupconsisting of leucine, methionine, and homoleucine. In a preferredembodiment, R₂ is OR, SR, a C₃-C₆ alkyl, an aryl, or a side chain of anamino acid selected from the group consisting of norleucine, methionine,leucine, and homoleucine. In a preferred embodiment, R₄ is a side chainof an amino acid selected from the group consisting of glutamine,asparagine, and homoserine. In a preferred embodiment, R₅ is a sidechain of an amino acid selected from the group consisting of alanine,valine, and serine. In a preferred embodiment, Y is OH, OR′, NHR′, NR′₂,or NH₂. Combinations of these embodiments are also contemplated. Infurther preferred embodiments, R₁, R₂, R₄, and R₅ are amino acid sidechains as shown in Table 3 below.

TABLE 3 Exemplary Embodiments of Formula IC Emb. R₁ R₂ R₄ R₅ A Leu NleGln Val, Ala, or Leu B Met Met Gln Val, Ala, or Leu C Hle Hle Gln Val,Ala, or Leu D Hle Leu Gln Val, Ala, or Leu E Met Leu Gln Val, Ala, orLeu F Leu Hle Gln Val, Ala, or Leu G Hle Nle Gln Val, Ala, or Leu

Exemplary oxopiperazine compounds of the present invention include,without limitation, OHMs 23-25 and 28-31. In a preferred embodiment, theoxopiperazine is has a Rosetta score below 7 R.E.U. In a preferredembodiment, the oxopoperizine is selected from the group consisting ofOHM 23, OHM 25, OHM 30, and OHM 31.

Oxopiperazines of the present invention may be made using methods in theart. Suitable methods include those described in U.S. patent applicationSer. No. 12/917,176, which is hereby incorporated by reference in itsentirety.

Also encompassed by the present invention is a pharmaceuticalformulation that includes an oxopiperazine of the present invention anda pharmaceutically acceptable vehicle.

Suitable pharmaceutical formulations include the oxopiperazine and anypharmaceutically acceptable adjuvants, carriers, solutions, suspensions,emulsions, excipients, powders, and/or stabilizers, and can be in solidor liquid form, such as tablets, capsules, powders, solutions,suspensions, or emulsions. The compositions preferably contain fromabout 0.01 to about 99 weight percent, more preferably from about 2 toabout 60 weight percent, of the oxopiperazine together with theadjuvants, carriers and/or excipients. The amount of active compound insuch therapeutically useful compositions is such that a suitable dosageunit will be obtained.

In addition, the pharmaceutical formulations of the present inventionmay further comprise one or more pharmaceutically acceptable diluents orvehicles, such as preserving agents, fillers, disintegrating agents,wetting agents, emulsifying agents, suspending agents, sweeteningagents, flavoring agents, perfuming agents, antibacterial agents,antifungal agents, lubricating agents and dispensing agents, dependingon the nature of the mode of administration and dosage forms. Examplesof suspending agents include ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monosterate andgelatin. Examples of suitable carriers, diluents, solvents, or vehiclesinclude water, ethanol, polyols, suitable mixtures thereof, vegetableoils (such as olive oil), and injectable organic esters such as ethyloleate. Examples of excipients include lactose, milk sugar, sodiumcitrate, calcium carbonate, and dicalcium phosphate. Examples ofdisintegrating agents include starch, alginic acids, and certain complexsilicates. Examples of lubricants include magnesium stearate, sodiumlauryl sulphate, talc, as well as high molecular weight polyethyleneglycols.

For oral therapeutic administration, the oxopiperazines of the inventionmay be incorporated with excipients and used in the form of tablets,capsules, elixirs, suspensions, syrups, and the like. Such compositionsand preparations should contain at least 0.1% of the oxopiperazine. Thepercentage of the oxopiperazine in these compositions may, of course, bevaried and may conveniently be between about 2% to about 60% of theweight of the unit. The amount of the oxopiperazine in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, or alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient(s), sucrose as a sweetening agent, methyl and propylparabensas preservatives, a dye, and flavoring such as cherry or orange flavor.

Solutions or suspensions of the oxopiperazine (for example, forparenteral administration) can be prepared in water suitably mixed witha surfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In allcases, the form must be sterile and must be fluid to the extent thateasy syringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), suitable mixtures thereof, and vegetable oils.

Another aspect of the present invention relates to inhibiting theHIF1α-p300/CBP interaction using the oxopiperazines of the presentinvention.

One embodiment of this aspect of the present invention relates to amethod of reducing transcription of a gene in a cell, wheretranscription of the gene is mediated by interaction of HIF1α withCREB-binding protein and/or p300. This method involves contacting thecell with an oxopiperazine of the present invention under conditionseffective to cause nuclear uptake of the oxopiperazine, where theoxopiperazine disrupts interaction of HIF1α and p300/CBP and therebyreduces transcription of the gene.

Genes whose transcription is mediated by interaction of HIF1α with CBPand/or p300 include α_(1B)-adrenergic receptor, adenylate kinase 3,adrenomedullin, aldolase A, aldolase C, carbonic anhydrase IX,ceruloplasmin, chemokine receptor type 4 (CXCR4, fusin, CD184), c-Met,endothelin-1, enolase 1, erythropoietin, glucose transporter 1, glucosetransporter 3, glyceraldehyde-3-phosphate dehydrogenase, heme oxygenase1, hexokinase 1, hexokinase 2, IGF binding protein 1, IGF bindingprotein 3, insulin-like growth factor 2, lactate dehydrogenase A, lysyloxidase, monoamine oxidase isoform A, monoamine oxidase isoform B,nitric oxide synthase 2, p21, p35^(srg), phosphofructokinase,phosphoglycerate kinase 1, plasminogen activator inhibitor 1, pyruvatekinase M, stromal-derived factor 1, tranferrin receptor, transferrin,transforming growth factor β₃, triose phosphate isomerase 1, vascularendothelial growth factor, vascular endothelial growth factor receptorFLT-1, and vascular endothelial growth factor receptor KDR/Flk-1. Someuses for inhibiting transcription of these genes are shown in Table 4.

TABLE 4 Example Disorders Gene Treat/prevent adrenomedullinPheochromocytoma carbonic anhydrase IX Cancer ceruloplasmin Lymphoma,acute and chronic inflammation, rheumatoid arthritis chemokine receptortype 4 Cancer stem cell migration, (CXCR4, fusin, CD184) inflammationc-Met Metastasis (tumor, incl. cancer) endothelin-1 Abnormalvasoconstriction enolase 1 Hashimoto's encephalopathy, severe asthmaerythropoietin Abnormal oxygen transport glucose transporter 1 Aerobicglycolysis (Warburg effect) glucose transporter 3 Aerobic glycolysis(Warburg effect) heme oxygenase 1 Abnormal oxygen transport hexokinase 1Aerobic glycolysis (Warburg effect) hexokinase 2 Aerobic glycolysis(Warburg effect) IGF binding protein 1 Abnormal development and functionof organs (brain, liver) IGF binding protein 3 Abnormal development andfunction of organs (brain, liver) insulin-like growth factor 2 Abnormaldevelopment and function of organs (brain, liver) lactate dehydrogenaseA Myocardial infarction lysyl oxidase Metastasis (tumor, esp. breastcancer) monoamine oxidase isoform A Aggression, depression, cancer, esp.prostate monoamine oxidase isoform B Aggression, depression, cancer,esp. prostate nitric oxide synthase 2 Abnormal vasomotor tonephosphofructokinase Aerobic glycolysis (Warburg effect) phosphoglyceratekinase 1 Aerobic glycolysis (Warburg effect) stromal-derived factor 1Cancer stem cell migration, inflammation tranferrin receptor Abnormaliron uptake/metabolism transferrin Abnormal iron uptake/metabolismtriose phosphate isomerase 1 Aerobic glycolysis (Warburg effect)vascular endothelial growth Angiogenesis (tumor, incl. cancer) factorvascular endothelial growth Angiogenesis (tumor, incl. cancer) factorreceptor FLT-1 vascular endothelial growth Angiogenesis (tumor, incl.cancer) factor receptor KDR/Flk-1

Another embodiment of this aspect of the present invention relates to amethod of treating or preventing in a subject a disorder mediated byinteraction of HIF1α with CBP and/or p300. This method involvesadministering an oxopiperazine of the present invention to the subjectunder conditions effective to treat or prevent the disorder.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder mediated by interaction of HIF1α with CBP and/orp300. This is because the oxopiperazines are expected to act asinhibitors of HIF1α binding to CBP and/or p300. As used herein, the term“treatment” is defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease, a symptom of disease, or a predisposition toward adisease, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve, or affect the disease, the symptoms ofdisease, or the predisposition toward disease.

Disorders that can be treated or prevented include, for example, retinalischemia (Zhu et al., Invest. Ophthalmol. Vis. Sci. 48:1735-43 (2007);Ding et al., Invest. Ophthalmol. Vis. Sci. 46:1010-6 (2005), each ofwhich is hereby incorporated by reference in its entirety), pulmonaryhypertension (Simon et al., Annu. Rev. Physiol. 70:51-71 (2008); Eul etal., FASEB J. 20:163-5 (2006), each of which is hereby incorporated byreference in its entirety), intrauterine growth retardation (Caramelo etal., Medicina B. Aires 66:155-64 (2006); Tazuke et al., Proc. Nat'lAcad. Sci. USA 95:10188-93 (1998), each of which is hereby incorporatedby reference in its entirety), diabetic retinopathy (Ritter et al., J.Clin. Invest. 116:3266-76 (2006); Wilkinson-Berka et al., Curr. Med.Chem. 13:3307-17 (2006); Vinores et al., J. Cell. Physiol. 206:749-58(2006); Caldwell et al., Curr. Drug Targets 6:511-24 (2005), each ofwhich is hereby incorporated by reference in its entirety), age-relatedmacular degeneration (Inoue et al., Br. J. Ophthalmol. 91:1720-1 (2007);Zuluaga et al., Invest. Ophthalmol. Vis. Sci. 48:1767-72 (2007); Provis,Prog. Retin. Eye Res. 20:799-821 (2001), each of which is herebyincorporated by reference in its entirety), diabetic macular edema(Vinores et al., J. Cell. Physiol. 206:749-58 (2006); Forooghian & Das,Am. J. Ophthalmol. 144:761-8 (2007), each of which is herebyincorporated by reference in its entirety), and cancer (Marignol et al.,Cancer Treat. Rev. 34:313-27 (2008); Galanis et al., Cancer Lett.266:12-20 (2008); Ushio-Fukai & Nakamura, Cancer Lett. 266:37-52 (2008);Adamski et al., Cancer Treat. Rev. 34:231-46 (2008); Toffoli & Michiels,FEBS J. 275:2991-3002 (2008); Peehl & Coram, J. Urol. 180:2206-11(2008); Flamand & Zhao, J. Cancer Res. Clin. Oncol. 136:1761-71 (2010),each of which is hereby incorporated by reference in its entirety).

The subject according to this aspect of the present invention can be,for example, any vertebrate, e.g., mammals, fish, reptiles, birds, andamphibians. Suitable mammals include, for example, primates, felines,canines, rodents (e.g., mice and rats), and livestock (e.g., cattle,sheep, pigs, goats, and horses). In a preferred embodiment, the subjectis a human subject.

Yet another embodiment of this aspect of the present invention relatesto a method of reducing or preventing angiogenesis in a tissue. Thismethod involves contacting the tissue with an oxopiperazine of thepresent invention under conditions effective to reduce or preventangiogenesis in the tissue.

Another embodiment of this aspect of the present invention relates to amethod of inducing apoptosis of a cell. This method involves contactingthe cell with an oxopiperazine of the present invention under conditionseffective to induce apoptosis of the cell.

Another embodiment of this aspect of the present invention relates to amethod of decreasing survival and/or proliferation of a cell. Thismethod involves contacting the cell with an oxopiperazine of the presentinvention under conditions effective to decrease survival and/orproliferation of the cell.

Suitable cells according to the methods of the present inventioninclude, without limitation, any vertebrate cell, e.g., mammalian,ichthian, reptilian, avian, and amphibian cells. Suitable mammaliancells include, for example, those of primates, felines, canines, rodents(e.g., mice and rats), and livestock (e.g., cattle, sheep, pigs, goats,and horses). In a preferred embodiment, the cells are human cells.

Contacting (including administering) according to the methods of thepresent invention can be carried out using methods that will be apparentto the skilled artisan, and can be done in vitro or in vivo.

One approach for delivering agents into cells involves the use ofliposomes. Basically, this involves providing a liposome which includesagent(s) to be delivered, and then contacting the target cell, tissue,or organ with the liposomes under conditions effective for delivery ofthe agent into the cell, tissue, or organ.

This liposome delivery system can also be made to accumulate at a targetorgan, tissue, or cell via active targeting (e.g., by incorporating anantibody or hormone on the surface of the liposomal vehicle). This canbe achieved according to known methods.

An alternative approach for delivery of protein- orpolypeptide-containing agents (e.g., oxopiperazines of the presentinvention containing one or more protein or polypeptide side chains)involves the conjugation of the desired agent to a polymer that isstabilized to avoid enzymatic degradation of the conjugated protein orpolypeptide. Conjugated proteins or polypeptides of this type aredescribed in U.S. Pat. No. 5,681,811 to Ekwuribe, which is herebyincorporated by reference in its entirety.

Yet another approach for delivery of agents involves preparation ofchimeric agents according to U.S. Pat. No. 5,817,789 to Heartlein etal., which is hereby incorporated by reference in its entirety. Thechimeric agent can include a ligand domain and the agent (e.g., anoxopiperazine of the invention). The ligand domain is specific forreceptors located on a target cell. Thus, when the chimeric agent isdelivered intravenously or otherwise introduced into blood or lymph, thechimeric agent will adsorb to the targeted cell, and the targeted cellwill internalize the chimeric agent.

Oxopiperazines of the present invention may be delivered directly to thetargeted cell/tissue/organ.

Additionally and/or alternatively, the oxopiperazines may beadministered to a non-targeted area along with one or more agents thatfacilitate migration of the oxopiperazines to (and/or uptake by) atargeted tissue, organ, or cell. As will be apparent to one of ordinaryskill in the art, the oxopiperazine itself can be modified to facilitateits transport to a target tissue, organ, or cell, including itstransport across the blood-brain barrier; and/or to facilitate itsuptake by a target cell (e.g., its transport across cell membranes). Ina preferred embodiment, the oxopiperazine of the invention is modified,and/or delivered with an appropriate vehicle, to facilitate its deliveryto the nucleus of the target cell (Wender et al., Proc. Nat'l Acad. Sci.USA 97:13003-8 (2000); Roberts, Scientist 18:42-3 (2004); Joliot &Prochiantz, Nat. Cell Biol. 6:189-96 (2004), each of which is herebyincorporated by reference in its entirety). Some example target cells,tissues, and/or organs for the embodiments described above are shown inTable 5.

TABLE 5 Example Target Cells/Tissues/Organs Desired Effect ExampleTarget(s) Inhibit transcription of: adrenomedullin Pheochromocytomacarbonic anhydrase IX Tumor cells/tissue, incl. cancer ceruloplasminLymphocytes/lymphatic tissue, inflamed tissue, rheumatoid arthritictissue chemokine receptor type 4 Tumor cells/tissue, incl. cancer(CXCR4, fusin, CD184) c-Met Tumor cells/tissue, incl. cancerendothelin-1 Endothelium enolase 1 Liver, brain, kidney, spleen,adipose, lung erythropoietin Liver, kidney glucose transporter 1 Tumor,incl. cancer glucose transporter 3 Tumor, incl. cancer hexokinase 1Tumor, incl. cancer hexokinase 2 Tumor, incl. cancer IGF binding protein1 Brain, liver IGF binding protein 3 Brain, liver insulin-like growthfactor 2 Brain, liver lactate dehydrogenase A Heart lysyl oxidase Tumorcells/tissue, incl. cancer monoamine oxidase isoform A Tumorcells/tissue, esp. prostate cancer monoamine oxidase isoform B Tumorcells/tissue, esp. prostate cancer nitric oxide synthase 2 Vessels,cardiovascular cells/tissue phosphofructokinase Tumor, incl. cancerphosphoglycerate kinase 1 Tumor, incl. cancer stromal-derived factor 1Tumor cells/tissue, incl. cancer transferrin Liver triose phosphateisomerase 1 Tumor, incl. cancer vascular endothelial growth Tumorcells/tissue, incl. cancer factor (VEGF) VEGF receptor FLT-1 Tumorcells/tissue, incl. cancer VEGF receptor KDR/Flk-1 Tumor cells/tissue,incl. cancer Treat or prevent: retinal ischemia Retina (eye) pulmonaryhypertension Lungs intrauterine growth retardation Uterus diabeticretinopathy Retina (eye) age-related macular degeneration Retina (eye)diabetic macular edema Retina (eye) Reduce or prevent angiogenesis Tumorcells/tissue, incl. cancer Reduce or prevent metastasis Tumorcells/tissue, incl. cancer Decrease cell survival and/ Cancerous cells,cells contained in the or proliferation endothelial vasculature of atissue that contains cancerous cells

In vivo administration can be accomplished either via systemicadministration to the subject or via targeted administration to affectedtissues, organs, and/or cells, as described above. Typically, thetherapeutic agent (i.e., an oxopiperazine of the present invention) willbe administered to a patient in a vehicle that delivers the therapeuticagent(s) to the target cell, tissue, or organ. Typically, thetherapeutic agent will be administered as a pharmaceutical formulation,such as those described above.

Exemplary routes of administration include, without limitation, orally,topically, transdermally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, intraventricularly, andintralesionally; by intratracheal inoculation, aspiration, airwayinstillation, aerosolization, nebulization, intranasal instillation,oral or nasogastric instillation, intraperitoneal injection,intravascular injection, intravenous injection, intra-arterial injection(such as via the pulmonary artery), intramuscular injection, andintrapleural instillation; by application to mucous membranes (such asthat of the nose, throat, bronchial tubes, genitals, and/or anus); andby implantation of a sustained release vehicle.

For use as aerosols, an oxopiperazine of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The oxopiperazines of the present invention also may beadministered in a non-pressurized form.

Exemplary delivery devices include, without limitation, nebulizers,atomizers, liposomes (including both active and passive drug deliverytechniques) (Wang & Huang, Proc. Nat'l Acad. Sci. USA 84:7851-5 (1987);Bangham et al., J. Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda; and U.S.Pat. No. 5,059,421 to Loughrey et al.; Wolff et al., Biochim. Biophys.Acta 802:259-73 (1984), each of which is hereby incorporated byreference in its entirety), transdermal patches, implants, implantableor injectable protein depot compositions, and syringes. Other deliverysystems which are known to those of skill in the art can also beemployed to achieve the desired delivery of the oxopiperazine to thedesired organ, tissue, or cells in vivo to effect this aspect of thepresent invention.

Contacting (including in vivo administration) can be carried out asfrequently as required and for a duration that is suitable to providethe desired effect. For example, contacting can be carried out once ormultiple times, and in vivo administration can be carried out with asingle sustained-release dosage formulation or with multiple (e.g.,daily) doses.

The amount to be administered will, of course, vary depending upon theparticular conditions and treatment regimen. The amount/dose required toobtain the desired effect may vary depending on the agent, formulation,cell type, culture conditions (for ex vivo embodiments), the durationfor which treatment is desired, and, for in vivo embodiments, theindividual to whom the agent is administered.

Effective amounts can be determined empirically by those of skill in theart. For example, this may involve assays in which varying amounts ofthe oxopiperazine of the invention are administered to cells in cultureand the concentration effective for obtaining the desired result iscalculated. Determination of effective amounts for in vivoadministration may also involve in vitro assays in which varying dosesof agent are administered to cells in culture and the concentration ofagent effective for achieving the desired result is determined in orderto calculate the concentration required in vivo. Effective amounts mayalso be based on in vivo animal studies.

The present invention may be further illustrated by reference to thefollowing examples.

EXAMPLES

The following Examples are intended to illustrate, but by no means areintended to limit, the scope of the present invention as set forth inthe appended claims.

Example 1—General Materials and Methods

Commercial-grade reagents and solvents were used without furtherpurification except as indicated. All reactions were stirredmagnetically or mechanically shaken; moisture-sensitive reactions wereperformed under nitrogen atmosphere. Reverse-phase HPLC experiments wereconducted with 0.1% aqueous trifluoroacetic acid and 0.1%trifluoroacetic acid in acetonitrile buffers as eluents on C₁₈reversed-phase columns using a Beckman Coulter HPLC equipped with aSystem Gold 168 Diode array detector. ESIMS data was obtained on anAgilent 1100 series LC/MSD (XCT) electrospray trap. The microwavereactions were performed in the CEM Discover single-mode reactor withcontrolled power, temperature, and time settings. The NMR spectra ofoxopiperazine compounds were recorded on a Bruker AVANCE 400, 500, or600 MHz spectrometer.

Example 2—p300-CH1 Plasmids

The DNA sequence of the human p300 CH1 domain (amino acid residues323-423) was subcloned into a pUC57 plasmid by Genscript, Inc. Aftertransformation of JM109 bacteria (Promega) with the plasmid, it wasamplified and purified. Then the gene of interest was subcloned betweenBamHI and EcoRI restriction sites of pGEX-4T-2 expression vector(Amersham).

Example 3—p300-CH1 Protein Expression and Purification

The pGEX-4T-2-p300 fusion vector was transformed into BL21(DE3)competent E.coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as theprimary nitrogen source. Protein production was induced with 1 mM IPTGat OD₆₀₀ of 1 for 16 hours at 15° C. Production of the desiredp300-CH1-GST fusion product was verified by SDS-PAGE. Bacteria wereharvested and resuspended in the lysis buffer with 20 mM Phosphatebuffer (Research Products International, Corp.), 100 μM DTT (Fisher),100 μM ZnSO₄ (Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A(Research Products International, Corp.), 10 mg/mL Leupeptin A (ResearchProducts International, Corp.), 500 μM PMSF (Sigma), and 0.5% glycerolat pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. and20,000 rpm for 20 minutes. Fusion protein was collected from thebacterial supernatant and purified by affinity chromatography usingglutathione Sepharose 4B beads (Amersham) prepared according to themanufacturer's directions. GST-tag was cleaved by thrombin and proteinwas eluted from resin. Collected fractions were assayed by SDS-PAGE gel;pooled fractions were treated with protease inhibitor cocktail (Sigma)and against a buffer containing 10 mM Tris, 50 mM NaCl, 2 mM DTT(Fisher), and 3 equivalents ZnSO₄ at pH 8.0 to ensure proper folding(vide supra).

Example 4—Docking and Design Protocol in Rosetta

The oxopiperazine dimer scaffold was initially docked by aligning Cβatoms on the scaffold positions corresponding to hotspot residues onHIF1α (R₁: Leu818, R₂: Leu822, R₄: Gln824) using the PDB structure:1L8C. The Rosetta relax w/constraints application was run on thisinitial structure to relieve any clashes that may hinder score analysis.The relaxed complex was then modeled and designed using a protocoldeveloped specifically for oxopiperazine inhibitors. The protocoliterates between 1) a perturbation phase (conformational optimization),attempting to find the lowest energy conformation of bound ligand andtarget protein given the current residue identities; and 2) a designphase, which attempts to find residue substitutions includingnoncanonical analogues that lower the energy given the currentconformation. The perturbation phase consists of a) rigid body rotationand translation moves, b) small angle moves of phi and psi, and c)pucker moves of the oxopiperazine rings. Perturbations were only allowedto the scaffold, leaving the target's backbone fixed. All residues atthe interface on both target and ligand were allowed to sampleside-chain rotamer space. The design phase consisted of residue identitysubstitutions at positions along the scaffold and rotamer repacking.Substitutions were defined in the Rosetta resfile. Finally, minimizationof all degrees of freedom in the complex was performed.

For modeling analysis, the same design protocol was used, exceptresidues were fixed to the identities of interest in the Rosetta residueinput file (i.e., resfile). Fixing residue identities only allows sidechain optimization during the “design” phase. 5000 independent runs(i.e., decoys) were computed for each sequence. For design runs, R₁ andR₂ were substituted with all hydrophobic noncanonical amino acids inTable 6 below except for proline analogs.

TABLE 6 Noncanonical Amino Acid Design Library for Rosetta (p300 Target)Positions R₁ and R₂ Rosetta code: noncanonical amino acid name NC A04:1-amino-cyclopentane-carboxylic_acid_puck1 NC A05:1-amino-cyclopentane-carboxylic_acid_puck2 NC A12:2.4-dimethyl-phenylalanine NC A20: 2-allyl-glycine NC A24:2-amino-2-phenylbutyric_acid NC A30: 2-amino-4-bromo-4-pentenoic_acid NCA31: 2-amino-5-phenyl-pentanoic_acid NC A33: 2-amino-heptanoic_acid NCA34: 2-aminomethyl-phenylalanine NC A44: 2-indanyl-glycine_puck1 NC A45:2-indanyl-glycine_puck2 NC A48: 2-methyl-phenylalanine NC A84:3-methyl-phenylalanine NC A91: 4.5-dehydro-leucine NC B04:4-amino-tetrahydrothiopyran-4-carboxylic_acid_boat1 NC B05:4-amino-tetrahydrothiopyran-4-carboxylic_acid_boat2 NC B06:4-amino-tetrahydrothiopyran-4-carboxylic_acid_chair1 NC B07:4-amino-tetrahydrothiopyran-4-carboxylic_acid_chair2 NC C90:4-fluoro-tryptophan NC B27: 4-methyl-phenylalanine NC B28:4-methyl-tryptophan NC B30: 4-phenyl-phenylalanine NC B31:4-tert-butyl-phenylalanine NC C80: 5-bromo-tryptophan NC C81:5-chloro-tryptophan NC B35: 5-fluoro-tryptophan NC B38:5-methyl-tryptophan NC C83: 6-bromo-tryptophan NC C84:6-chloro-tryptophan NC C85: 6-fluoro-tryptophan NC B40:6-methyl-tryptophan NC C86: 7-azatryptophan NC C87: 7-bromo-tryptophanNC C88: 7-methyl-tryptophan NC B44: 9-anthryl-alanine NC ABA:aminobutyric acid NC B47: allo-isoleucine NC B57: alpha-methyl-leucineNC B58: alpha-methyl-phenylalanine NC B60: alpha-methyl-tryptophan NCB62: alpha-methyl-valine NC B67: beta-(1-naphthyl)-alanine NC B74:beta-(2-naphthyl)-alanine NC B92:beta-beta-dicyclohexyl-alanine_boat_boat NC B93:beta-beta-dicyclohexyl-alanine_boat_chair NC B94:beta-beta-dicyclohexyl-alanine_chair_boat NC B95:beta-beta-dicyclohexyl-alanine_chair_chair NC B96:beta.beta-diphenyl-alanine NC B97: beta-chloro-alanine NC B99:beta-cyclohexyl-alanine_boat NC C00: beta-cyclohexyl-alanine_chair NCC01: beta-cyclopentyl-alanine NC C02: beta-cyclopentyl-alanine_puck NCC03: beta-fluoro-alanine NC C05: beta-iodo-alanine NC C11:cyclohexyl-glycine_boat NC C12: cyclohexyl-glycine_chair NC C15:diphenylglycine NC C16: dipropyl-glycine NC C20: ethionine NC C91:fluoro-leucine_ent1 NC C92: fluoro-leucine_ent2 NC C93:hexafluoro-leucine NC HLU: homoleucine NC C26: homocysteine NC C27:homophenylalanine NC MAL: MAL NC C12: MPA NC C36: n-in-methyl-tryptophanNC NLU: norleucine NC NVL: norvaline NC C41: penicillamine NC C42:phenylglycine NC C53: tert-butyl-alanine NC C54: tert-butyl-cysteine NCC55: tert-butyl-glycine NC C60: trifluoro-alanine NC C61:trifluoro-leucine NC C94: trifluoro-leucine_ent2

Detailed protocols including command lines have been previouslydescribed (Drew et al., PLoS One 8:DOI:10.1371/journal.pone.0067051(2013), which is hereby incorporated by reference in its entirety).

Top designs were selected based on filtering the lowest 5% of totalenergy decoys and sorting by Rosetta binding energy score. The Rosettabinding energy score was calculated using equation (1).Binding_energy_score=total_score−unbound_score  (1)

The unbound score was calculated by separating the scaffold from thetarget HIF1α structure, then repacking the side chains and finallycalculating the total Rosetta energy of the unbound complex.

Example 5—Rosetta Binding Discrimination Analysis

A random set of designs against target protein p300 were generated froma set of 30,000 Rosetta design runs where all four positions of anoxopiperazine dimer were allowed to vary to any canonical amino acidexcluding Cys, Gly, and Pro. The top 1000 of models by total Rosettascore made up the total random set. This random set is shown as a greyhistogram (violin plot) in FIG. 5.

The top binding energy score for designs with experimental bindingaffinities were determined from a set of 5,000 decoy structures. Asdescribed above, the top 1000 of decoys by total score was then sortedby Rosetta binding energy score and the lowest Rosetta binding energyscore was used.

Example 6—Quantum Mechanics Calculations

Quantum mechanics calculations were done using the Gaussian 09(EM64L-G09RevC.01, version date: 2011-09-23) software package (Gaussian09, Revision C.01, Frisch et al. (Gaussian, Inc. 2009), which is herebyincorporated by reference in its entirety). An initial optimizationusing “HF 6-31G(d) Opt SCRF=PCM SCF=Tight” parameters was done for eachmodel structure. The resulting optimized structure was then used forfurther energy calculations with parameters “B3LYP 6-31G(d) Geom=CheckSCRF=PCM SCF=Tight” and “MP2(full) 6-31G(d) Geom=Check SCRF=PCMSCF=Tight”.

Example 7—Synthesis and Characterization of Oxopiperazines

The oxopiperazines were synthesized via solid phase synthesis asdescribed in U.S. patent application Ser. No. 12/917,176 to Arora etal., which is hereby incorporated by reference in its entirety, as shownin Scheme 1 below.

An Fmoc amino acid linked to Wang or Knorr Rink Amide resin (I) wasextended to a dipeptide using standard Fmoc solid phase peptidesynthesis methods in a solid phase reaction vessel (Coin et al., Nat.Protoc. 2:3247 (2007), which is hereby incorporated by reference in itsentirety). The resultant dipeptide was deprotected with 20%piperidine/dimethylformamide (DMF) and the resin was washed sequentiallywith DMF, dichloromethane (DCM), methanol (MeOH), and diethyl ether anddried under vacuum. o-Nitrobenzenesulfonyl chloride (Ns-Cl, 10 eq) andcollidine (10 eq) were dissolved in dry DCM and added to the reactionvessel. The mixture was shaken for 2 hours at 23° C. to obtain compoundII.

The resin was washed sequentially with DMF, DCM, MeOH, and diethyl etherand dried for 12 hours under vacuum. The resin was transferred to aglass microwave tube (CEM). Triphenylphosphine (PPh₃, 10 eq) was addedand the tube was flushed with nitrogen gas for 30 minutes.Tetrahydrofuran (THF), diisopropyl azodicarboxylate (DIAD, 10 eq), and2-bromoethanol (10 eq) were added and the reaction mixture was subjectedto microwave irradiation (200 watts, 250 psi) for 10 minutes at 100° C.The resin was washed sequentially with THF, DMF, and DCM. The resin wastransferred to a solid phase vessel and treated with1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in THF for 2 hours. The resinwas washed with THF, DMF, DCM, and diethyl ether and allowed to dry for30 minutes followed by treatment with DBU and 2-mercaptoethanol in DMFfor 2 hours to obtain compound III.

Compound III was then washed with DMF, DCM, MeOH, and diethyl either anddried. The desired pre-activated Fmoc-amino acid was added to the resinand the mixture was shaken at 23° C. for 12 hours affording compound IV.

Nosyl protection and the ring formation steps were repeated to obtainoxopiperazine dimer V after cleavage from the resin with 95%trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane(TIPS).

Oxopiperazines 21-25 were synthesized and characterized by HPLC and¹H-NMR. Their structure and ¹H-NMR characterization values are shown inTable 7 below. HPLC traces are shown in FIGS. 6A-E. Oxopiperazines 26and 27 were synthesized and characterized as described in InternationalApplication No. PCT/US13/26722, which is hereby incorporated byreference in its entirety).

TABLE 7 Compound Characterization

Oxopiperazine 21: LLAQ-NH₂ ¹H-NMR (600 MHz, d₆-DMSO, 100° C.) δ 6.87(br, 3H), 6.61 (br, 3H), 5.36 (t, J = 7.47, 1H), 4.90-4.85 (m, 1H), 4.67(q, J = 6.88, 1H), 3.96 (br, 2H), 3.63- 3.24 (m, 8H), 1.95-1.82 (m, 3H),1.72-1.58 (m, 3H), 1.57-1.49 (m, 1H), 1.38 (s, 3H), 1.06-0.75 (m, 12H).HRMS (ESI) C₂₄H₄₂N₆O₅ [M + H]⁺ calc'd = 494.3217; found = 495.3502. SeeFIG. 6A.

Oxopiperazine 22: LAAQ ¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ 6.77 (br,2H), 6.52 (br, 2H), 5.34 (q, J = 6.91, 1H), 4.90-4.84 (m, 1H), 4.67 (q,J = 6.91, 1H), 4.01-3.91 (m, 1H), 3.76-3.69 (m, 1H), 3.57-3.27 (m, 7H),3.24-3.14 (m, 1H), 2.19-2.01 (m, 3H), 2.00-1.80 (m, 3H), 1.64-1.55 (m,1H), 1.38 (d, J = 6.92, 3H), 1.27 (d, J = 6.92, 3H), 0.94 (t, J = 6.02,6H). HRMS (ESI) C₂₁H₃₆N₆O₅ [M + H]⁺ calc'd = 453.2747; found = 453.2863.See FIG. 6B.

Oxopiperazine 23: L(Nle)AQ ¹H-NMR (600 MHz, d₆-DMSO, 100° C.) δ 6.87(br, 4H), 5.27 (t, J = 7.25, 1H), 4.92-4.83 (m, 1H), 4.67 (q, J = 7.07,1H), 3.96 (br, 1H), 3.88 (br, 1H), 3.59- 3.56 (m, 1H), 3.54-3.46 (m,3H), 3.45-3.39 (m, 2H), 3.36-3.31 (m, 1H), 3.30-3.21 (m, 1H), 2.12- 2.01(m, 2H), 1.99-1.83 (m, 2H), 1.80-1.59 (m, 4H), 1.39-1.20 (m, 8H), 0.95(d, J = 6.29, 3H), 0.94 (d, J = 6.19, 3H), 0.89 (t, J = 7.31, 3H). HRMS(ESI) C₂₄H₄₂N₆O₅ [M + H]⁺ calc'd = 495.3217; found = 495.3377. See FIG.6C.

Oxopiperazine 24: MMAQ ¹H-NMR (600 MHz, d₆-DMSO, 100° C.) δ 9.33 (br,1H), 6.90 (br, 3H), 5.46 (t, J = 6.91, 1H), 4.91-4.84 (m, 1H), 4.70-4.63(m, 1H), 4.16-3.91 (m, 1H), 3.87-3.75 (m, 1H), 3.59-3.54 (m, 2H), 3.50(t, J = 5.98, 2H), 3.47-3.40 (m, 1H), 3.36-3.22 (m, 3H), 2.70-2.60 (m,3H), 2.23-2.14 (m, 1H), 2.08 (d, J = 2.96, 3H), 2.07-1.87 (m, 5H),1.74-1.62 (m, 5H), 1.7 (br, 1H), 1.27 (s, 3H). HRMS (ESI) C₂₂H₃₈N₆O₅S₂[M + H]⁺ calc'd = 530.2345; found = 531.2423. See FIG. 6D.

Oxopiperazine 25: (Hle)(Hle)AQ ¹H-NMR (600 MHz, d₆-DMSO, 100° C.) δ 6.83(br, 4H), 5.26 (t, J = 7.06, 1H), 4.92-4.83 (m, 1H), 4.66 (q, J = 6.80,1H), 3.96 (br, 1H), 3.88 (br, 1H), 3.55- 3.46 (m, 3H), 3.45-3.36 (m,3H), 3.35-3.30 (m, 1H), 3.29-3.22 (m, 1H), 3.21 (s, 1H), 1.99-1.83 (m,2H), 1.83-1.63 (m, 3H), 1.62-1.49 (m, 2H), 1.46- 1.20 (m, 7H), 1.19-1.09(m, 2H), 0.95-0.83 (m, 12H). HRMS (ESI) C₂₄H₄₂N₅O₅ [M + H]⁺ calc'd =523.3530; found = 523.3642. See FIG. 6E.

Example 8—Tryptophan Fluorescence Binding Assay

Relative affinities of OHMs for p300-CH1 were determined using atryptophan fluorescence binding assay. Spectra were recorded on aQuantaMaster 40 spectrofluorometer (Photon Technology International) ina 10 mm quartz fluorometer cell at 25° C. with 4 nm excitation and 4 nmemission slit widths from 200 to 400 nm at intervals of 1 nm/s. Sampleswere excited at 295 nm and fluorescence emission was measured from200-400 nm and recorded at 335 nm. OHM stock solutions were prepared inDMSO. Aliquots containing 1 μL DMSO stocks were added to 400 μL of 1 μMp300-CH1 in 50 mM Tris and 100 μM NaCl (pH 8.0). After each addition,the sample was allowed to equilibrate for 5 minutes before UV analysis.Background absorbance and sample dilution effects were corrected bytitrating DMSO into p300-CH1 in an analogous manner. Final fluorescenceis reported as the absolute value of [(F₁-F₀)/F₁]*100, where F₁ is thefinal fluorescence upon titration, and F₀ is the fluorescence of theblank DMSO titration. EC₅₀ values for each peptide were determined byfitting the experimental data to a sigmoidal dose-response nonlinearregression model in GraphPad Prism 5.0, and the dissociation constants,K_(D), were obtained from equation (2).K _(D)=(R _(T)×(1−F _(SB))+L _(ST) ×F _(SB) ²)/F _(SB) −L _(ST)  (2)

where:

-   -   R_(T)=Total concentration of p300-CH1    -   L_(ST)=Total concentration of fluorescent peptide    -   F_(SB)=Fraction of bound fluorescent peptide        Results and Discussion of Examples 1-8

Protein design is the process of predicting an amino acid sequence thatwill fold into a desired structure or carry out a desired function(Butterfoss & Kuhlman, Ann. Rev. Biophys. Biomolec. Struct. 35:49(2006), which is hereby incorporated by reference in its entirety). Inorder to design more potent analogs, a computational approach thatcombines success in computational protein design (Butterfoss & Kuhlman,Ann. Rev. Biophys. Biomolec. Struct. 35:49 (2006); Kuhlman et al.,Science 302:1364 (2003); Jiang et al., Science 319:1387 (2008), each ofwhich is hereby incorporated by reference in its entirety) withpeptidomimetic scaffolds was used. Protein design principles to optimizethe affinity of oxopiperazine mimetics was achieved using Rosetta(rosettacommons.org) (Leaver-Fay et al., Methods Enzymol. 487:545(2011), which is hereby incorporated by reference in its entirety).

The objective of computational molecular design is to reduce the totalnumber of possible designs to a manageable number that can beefficiently synthesized and experimentally tested. An oxopiperazinedimer has four variable positions and assuming a standard library of 17amino acids (20 canonical amino acids without Cys, Gly, and Pro), thetotal number of possible designs would be >83,500. This calculation doesnot account for noncanonical amino acids, whose inclusion significantlyraises the number of potential designs. Experimentally synthesizing andtesting this many designs would be prohibitive for academic labs.Rosetta computational design reduces the number of total designs onemust synthesize to obtain potent ligands and streamlines the process offinding a high affinity binder.

A Rosetta oxopiperazine design protocol was recently used to designoxopiperazine helix mimetics that target the p53-Mdm2 interaction (U.S.Provisional Patent Application No. 61/979,784 to Arora et al., which ishereby incorporated by reference in its entirety). To further validatethe potential of the Rosetta oxopiperazine design protocol, inhibitorsof a different transcriptional complex were developed. It has recentlybeen shown that stabilized peptide helices (Kushal et al., Proc. Nat'lAcad. Sci. U.S.A. 110:15602 (2013), which is hereby incorporated byreference in its entirety) and small molecule oxopiperazine analogs (Lauet al., Proc. Nat'l Acad. Sci. 10.1073/pnas.1402393111 (published onlineMay 14, 2014), which is hereby incorporated by reference in itsentirety) that mimic a key helical domain of HIF1α can inhibit hypoxiainducible signaling in cell culture and animal models. The C-terminaldomain of HIF1α utilizes two short α-helices to bind to the CH1 domainof p300/CBP (FIG. 7). Computational alanine scanning (Kortemme et al.,Nat. Struct. Mol. Biol. 11:371 (2004), which is hereby incorporated byreference in its entirety) studies on the complex reveal that fourhelical residues from the HIF1α helix₈₁₆ ₈₂₄ (Leu818, Leu822, Asp823,and Gln824) make close contacts with the CH1 domain of p300/CBP. Threeof these residues, Leu818, Leu822 and Gln824, can be mimicked byoxopiperazine dimers consisting of the appropriate building blocks (FIG.7). Based on this analysis, analogs of HIF1α were designed to inhibitits binding with p300/CBP. The design and p300/CPB binding properties ofoxopiperazine helix mimetics (OHMs) 21-31 are shown in Table 8 below.

TABLE 8 Oxopiperazine HIF Mimics Targeting the CH1 Domain of p300/CBP-Rosetta Predicted Binding Energy vs. Experimental K_(d) (nM)

OHM R₁ R₂ R₃ R₄ Kd (μM)^(a) R.E.U.^(b) 21 Leu Leu Ala Gln 0.53 ± 0.1410.1 22 Leu Ala Ala Gln >10 11.4 23 Leu Nle Ala Gln 0.03 ± 0.01 5.34 24Met Met Ala Gln 0.24 ± 0.04 10.3 25 Hle Hle Ala Gln 0.16 ± 0.06 5.80 26Leu Leu Ala Ala 0.62 ± 0.27 10.2 27 Ala Ala Ala Ala >10 11.7 28 Hle LeuAla Gln ND^(c) 10.4 29 Met Leu Ala Gln ND^(c) 11.9 30 Leu Hle Ala GlnND^(c) 6.42 31 Hle Nle Ala Gln ND^(c) 9.63 ^(a)Binding affinity forp300-CH1 was determined using an intrinsic tryptophan fluorescenceassay. ^(b)Rosetta predicted binding energy. ^(c)ND = Not determined.OHMs 28-31 were not synthesized, but are expected to bind to p300/CPB aswell as or better than OHM 21.

OHM 21 contains projections representing all three wild-type residuesfrom HIF1α: R₁ as Leu818, R₂ as Leu822, and R₄ as Gln824 (Table 8). TheR₃ position of the oxopiperazine scaffold was not predicted to makecontacts with the target protein; an alanine residue was inserted atthis position. OHM 22 was designed as a single mutant of OHM 21 with theR₂ position substituted with an alanine residue.

Results with OHMs 21 and 22 have been previously described (Lau et al.,Proc. Nat'l Acad. Sci. 10.1073/pnas.1402393111 (published online May 14,2014); International Application No. PCT/US13/26722 to Arora et al.;Bullock Lau et al., Proc. Nat'l Acad. Sci. 10.1073/pnas.1402393111(published online May 14, 2014), each of which is hereby incorporated byreference in its entirety)). Oxopiperazine 21, consisting of thewild-type residues, bound the CH1 domain of p300 with an affinity of533±24 nM; whereas, the negative control OHM 22 displayed a very weakaffinity for p300-CH1, with a K_(d) value of >30 μM (Table 8 and FIG.8). The binding affinities of OHMs 21-25 for p300-CH1 were evaluatedusing intrinsic tryptophan fluorescence spectroscopy (Kushal et al.,Proc. Nat'l Acad. Sci. U.S.A. 110:15602 (2013); Dial et al.,Biochemistry 42:9937 (2003), each of which is hereby incorporated byreference in its entirety). Because Trp403 lies in the binding cleft ofp300/CBP where a native HIF1α₈₁₆₋₈₂₄ helix binds, it offers a probe forinvestigating mimetics of this helix. As part of this earlier study, theinteraction of OHM 21 with the p300-CH1 domain was also characterizedusing ¹H-¹⁵N HSQC NMR titration experiments with the uniformly¹⁵N-labeled CH1. Addition of OHM 21 led to consistent shifts inresonances of residues corresponding to the HIF1α₈₁₆₋₈₂₄ binding pocket.OHM 21 efficiently downregulated HIF signaling in cell culture atmicromolar levels and reduced tumor levels in triple-negative breastcancer cell line MDA-MB-231 mouse xenograft models. Importantly,microarray gene expression profiling data showed that the designedoxopiperazine helix mimetic, despite its low molecular weight and alimited number of contacts with the intended target protein, shows highspecificity on a genome-wide scale.

The encouraging results with OHM 21 provided a platform to test thepotential of the Rosetta peptidomimetic design strategy to designfurther HIF mimetics with improved functionality. Whether thecomputational approach could rapidly suggest nonnatural residues thatmay boost the binding affinity for p300-CH1 was examined. The p300/OHM21 binding was analyzed using the established protocol, with a libraryof noncanonical amino acids (Table 6, supra). The computationalpredictions suggested that inclusion of longer aliphatic side chains inplace of the isobutyl group of leucine would lead to better contactswith the hydrophobic pocket. Specifically, substitution with nonnaturalside chains at the R₂ position of OHM 21 was predicted to lead to anoptimized binder (FIGS. 9A-D).

FIG. 5 shows a violin plot for OHMs targeting the CH1 domain. The grayarea represents the top 1000 scores from Rosetta's evaluation of 30,000designs. The predicted high affinity designs feature norleucine (Nle)and homoleucine (Hle) residues in place of the wild-type leucine analogsand are substantially lower in energy than the rest of the sequencestested by Rosetta. Substitution of the two leucine residues withmethionines was predicted to be less effective than with noncanonicalresidues, suggesting that space-filling and polarity of side chaingroups are necessary for optimal results. Other combinations ofhomoleucine, norleucine, and leucine residues were also examined (seeTable 8, supra).

To experimentally evaluate the predictions, three analogs were preparedrepresenting top designs in which both leucine groups of OHM 21 weresubstituted with methionine, norleucine, or homoleucine to obtain OHMs23-25 (Table 8, supra). Each of these compounds bound p300 with higheraffinity than the parent OHM 21, with OHM 23 providing a 13-foldenhancement in binding affinity (K_(d)=30.2±1.87 nM).

A strong correlation was found between the experimental results forp300/CBP and Rosetta predictions (FIG. 10 and Table 8), furtherhighlighting the success of the computational design protocol. Since thefluorescence-binding assay uses a native tryptophan residue in thetarget molecular pocket, it provides a stringent test for the bindingsite specificity. Characterization of the interaction of OHM 21 withp300-CH1 domain using ¹H-¹⁵N HSQC NMR titration experiments furtherconfirms the target pocket for the designed analogs.

Example 9—Cross-Specificities of the Designed Compounds for Mdm2 andp300

Analyses of protein-protein interaction (PPI) networks suggest that thehuman interactome consists of hundreds of thousands of different PPIs(Bonetta, Nature 468:851 (2010); Venkatesan et al., Nat. Meth. 6:83(2009), each of which is hereby incorporated by reference in itsentirety). Analysis of the high-resolution protein complexes availablein the Protein Data Bank reveals that up to 60% of such proteincomplexes contain an interfacial α-helix (Bullock et al., J. Am. Chem.Soc. 133:14220 (2011); Jochim & Arora, ACS Chem. Biol. 5:919 (2010);Jochim & Arora, Mol. BioSyst. 5:924 (2009), each of which is herebyincorporated by reference in its entirety). Thus, a central question inthe design of helix mimetics as PPI inhibitors pertains to theirspecificity on the genome-wide scale. The specificity of OHM 21,designed to be a transcriptional inhibitor for off-target regulation,was recently probed using the Affymetrix Human Gene ST 1.0 arrayscontaining oligonucleotide sequences representing over 28,000transcripts (Lau et al., Proc. Nat'l Acad. Sci. 10.1073/pnas.1402393111(published online May 14, 2014), which is hereby incorporated byreference in its entirety). This compound was found to be remarkablyspecific given the limited number of contacts it offers.

As described herein, potent small molecule helix mimetics that featurenoncanonical side chains have been computationally designed as potentialinhibitors of protein-protein interactions. Oxopiperazine mimics of p53that inhibit the p53-Mdm2 interaction are described in U.S. ProvisionalPatent Application No. 61/979,784 to Arora et al., which is herebyincorporated by reference in its entirety. As a preliminary analysis ofRosetta's ability to predict the specificity of OHMs against unintendedtargets, the binding affinity of HIF mimics OHM 23 and OHM 25 againstMdm2 (Table 9 and FIG. 11A), and that of p53 mimic OHM 18 (see FIG. 12)against p300-CH1 (Table 9 and FIG. 11B), were determined.

TABLE 9 Cross-Specificities of Oxopiperazine p53 and HIF Mimics Againstp300-CH1 and Mdm2. Ligand p300 K_(d) (μM)^(a) Mdm2 K_(d) (μM)^(b) 18 >300.3 ± 0.04 23 0.03 ± 0.01 >50 25 0.16 ± 0.06 >50 ^(a)Binding affinityfor p300-CH1 was determined using an intrinsic tryptophan fluorescenceassay. ^(b)Binding affinity for Mdm2 was determined using a competitivefluorescence polarization assay with Flu-p53 as a probe (see U.S.Provisional Application No. 61/979,784 to Arora et al., which is herebyincorporated by reference in its entirety).

These analogs were chosen because they represent the highest affinityligands obtained for their respective targets and contain noncanonicalresidues. Calculations with the modified version of Rosetta, describedabove, predict that the p53 mimic OHM 18 is a poor ligand for p300-CH1and that HIF mimetics OHM 23 and OHM 25 are not optimal designs forMdm2. Specifically, the calculated Rosetta binding energy (R.E.U.) forOHM 18/p300-CH1 binding is the same as that calculated for OHM 22, anegative control designed for the HIF/p300 interaction (FIG. 5).Likewise, Rosetta predicts compounds OHM 23 and OHM 25 to have ahigh-energy interaction with Mdm2; >6 R.E.U.'s when compared to OHM 18,the high affinity Mdm2 ligand (FIG. 13). These predictions wereconfirmed in experimental binding assays.

The binding of oxopiperazine derivatives was tested using the assaysdescribed above. As expected, the compounds are specific for theircognate receptors (Table 9, supra), with each showing more than 100-foldspecificity for the desired protein surface. These results providesupport for the hypotheses that the computational strategy describedherein can be used to ultimately predict specificity of the designedpeptidomimetics on the genome-wide scale.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed:
 1. An oxopiperazine selected from the group consistingof (i) Formula IA:

wherein: R₁ is hydrophobic and is an amino acid side chain, OR, SR, analkyl, or an aryl; wherein R is independently H, an alkyl, or an aryl;R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl; wherein Ris independently H, an alkyl, or an aryl; wherein R₂ is either a leucineside chain or R₂ is longer than a leucine side chain by at least onebackbone methylene; and with the proviso that R₁ and R₂ are not both anaryl; R₃ is hydrophobic and is an amino acid side chain, SR, or analkyl; wherein R is independently H, an alkyl, or an aryl; R₄ is ahydrogen bond donor, a hydrophobic amino acid side chain, or an amide;each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or an aryl;wherein each R is independently H, an alkyl, or an aryl; X₁ is H, N(R)₂,OR, COR′, CO₂R′, CONR′, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, a peptide of 1 to about 10 amino acid residues, a protectinggroup for protection of an amine, a solubilizing group, a targetingmoiety, or a tag; wherein each R is independently H, an alkyl, or anaryl; and wherein R′ is H, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; Z is N; A₁-W₁is:

and Y is OR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, an amino acid, a peptide of 1 to about 10 amino acidresidues, a protecting group for protection of a carboxylic acid, atargeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; andwherein each R′″ is independently H, CO₂R′, CONR′, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; and(ii) Formula IB:

wherein: R₀ is hydrophobic and is an amino acid side chain, OR, SR, analkyl, or an aryl; wherein R is independently H, an alkyl, or an aryl;R₁ and R₂ are each independently a solubilizing group, a hydrophobicamino acid side chain, H, N(R)₂, OR, SR, halogen, an alkyl, or an aryl;wherein each R is independently H, an alkyl, or an aryl; R₃ is an aminoacid side chain, OR, SR, an alkyl, or an aryl; wherein R isindependently H, an alkyl, or an aryl; wherein R₃ is either a leucineside chain or R₃ is longer than a leucine side chain by at least onebackbone methylene; and with the proviso that R₀ and R₃ are not both anaryl; R₄ is a hydrogen bond donor, a hydrophobic amino acid side chain,or an amide; each R₆ is independently H, N(R)₂, OR, halogen, an alkyl,or an aryl; wherein each R is independently H, an alkyl, or an aryl; X′is H, COR′, CO₂R′, CONR′, OR′, N(R″)₂, an alkyl, an aryl, an arylalkyl,a cycloalkyl, a heteroaryl, a solubilizing group, a targeting moiety, ora tag; wherein R′ is H, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, an amino acid residue, a peptide of 1 to about 10 amino acidresidues, a targeting moiety, or a tag; and wherein each R″ isindependently H, CO₂R′, CONR′, an alkyl, an aryl, an arylalkyl, acycloalkyl, a heteroaryl, a targeting moiety, or a tag; A₁-W₁ is:

and Y is OR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, an amino acid, a peptide of 1 to about 10 amino acidresidues, a protecting group for protection of a carboxylic acid, atargeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; andwherein each R′″ is independently H, CO₂R′, CONR′, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag;with the proviso that the oxopiperazine is not one of the followingcompounds


2. The oxopiperazine according to claim 1, wherein the oxopiperazine hasa formula of Formula IA.
 3. The oxopiperazine according to claim 2,wherein R₁ is OR, SR, a C₃-C₆ alkyl, an aryl, or a side chain of anamino acid selected from the group consisting of leucine, methionine,and homoleucine; R₂ is OR, SR, a C₃-C₆ alkyl, an aryl, or a side chainof an amino acid selected from the group consisting of norleucine,methionine, leucine, and homoleucine; R₃ is SR, a C₁-C₃ alkyl, or a sidechain of an amino acid selected from the group consisting of glycine andalanine; R₄ is a side chain of an amino acid selected from the groupconsisting of glutamine, alanine, valine, asparagine, serine, andhomoserine; and Y is OH, OR′, NEM′, NR′₂, or NH₂.
 4. The oxopiperazineaccording to claim 1, wherein the oxopiperazine has a formula of FormulaM.
 5. The oxopiperazine according to claim 4, wherein R₀ is OR, SR, aC₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected from thegroup consisting of leucine, methionine, and homoleucine; R₃ is OR, SR,a C₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected fromthe group consisting of norleucine, methionine, leucine, andhomoleucine; R₄ is a side chain of an amino acid selected from the groupconsisting of glutamine, alanine, valine, asparagine, serine, andhomoserine; and Y is OH, OR′, NHR′, NR′₂, or NH₂.
 6. The oxopiperazineaccording to claim 1, wherein the oxopiperazine is selected from thegroup consisting of oxopiperazine helix mimetic (“OHM”) 23, OHM 24, OHM25, OHM 28, OHM 29, OHM 30, and OHM 31:

OHM 23: R₁=Leu, R₂=Nle, R₃=Ala, R₄=Gln; OHM 24: R₁=Met, R₂=Met, R₃=Ala,R₄=Gln; OHM 25: R₁=Hle, R₂=Hle, R₃=Ala, R₄=Gln; OHM 28: R₁=Hle, R₂=Leu,R₃=Ala, R₄=Gln; OHM 29: R₁=Met, R₂=Leu, R₃=Ala, R₄=Gln; OHM 30: R₁=Leu,R₂=Hle, R₃=Ala, R₄=Gln; OHM 31: R₁=Hle, R₂=Nle, R₃=Ala, R₄=Gln.
 7. Theoxopiperazine according to claim 1, with the proviso that theoxopiperazine is not OHM 21:

OHM 21: R₁=Leu, R₂=Leu, R₃=Ala, R₄=Gln.
 8. A pharmaceutical formulationcomprising: an oxopiperazine according to claim 1 and a pharmaceuticallyacceptable vehicle.
 9. The pharmaceutical formulation according to claim8, wherein the oxopiperazine has a formula of Formula IA.
 10. Thepharmaceutical formulation according to claim 9, wherein R₁ is OR, SR, aC₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected from thegroup consisting of leucine, methionine, and homoleucine; R₂ is OR, SR,a C₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected fromthe group consisting of norleucine, methionine, leucine, andhomoleucine; R₃ is SR, a C₁-C₃ alkyl, or a side chain of an amino acidselected from the group consisting of glycine and alanine; R₄ is a sidechain of an amino acid selected from the group consisting of glutamine,alanine, valine, asparagine, serine, and homoserine; and Y is OH, OR′,NEM′, NR′₂, or NH₂.
 11. The pharmaceutical formulation according toclaim 8, wherein the oxopiperazine has a formula of Formula IB.
 12. Thepharmaceutical formulation according to claim 11, wherein R₀ is OR, SR,a C₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected fromthe group consisting of leucine, methionine, and homoleucine; R₃ is OR,SR, a C₃-C₆ alkyl, an aryl, or a side chain of an amino acid selectedfrom the group consisting of norleucine, methionine, leucine, andhomoleucine; R₄ is a side chain of an amino acid selected from the groupconsisting of glutamine, alanine, valine, asparagine, serine, andhomoserine; and Y is OH, OR′, NEM′, NR′₂, or NH₂.
 13. The pharmaceuticalformulation according to claim 8, wherein the oxopiperazine is selectedfrom the group consisting of oxopiperazine helix mimetic (“OHM”) 23, OHM24, OHM 25, OHM 28, OHM 29, OHM 30, and OHM 31:

OHM 23: R₁=Leu, R₂=Nle, R₃=Ala, R₄=Gln; OHM 24: R₁=Met, R₂=Met, R₃=Ala,R₄=Gln; OHM 25: R₁=Hle, R₂=Hle, R₃=Ala, R₄=Gln; OHM 28: R₁=Hle, R₂=Leu,R₃=Ala, R₄=Gln; OHM 29: R₁=Met, R₂=Leu, R₃=Ala, R₄=Gln; OHM 30: R₁=Leu,R₂=Hle, R₃=Ala, R₄=Gln; OHM 31: R₁=Hle, R₂=Nle, R₃=Ala, R₄=Gin.
 14. Thepharmaceutical formulation according to claim 8, with the proviso thatthe oxopiperazine is not OHM 21:

OHM 21: R₁=Leu, R₂=Leu, R₃=Ala, R₄=Gln.
 15. A method of reducingtranscription of a gene in a cell, wherein transcription of the gene ismediated by interaction of Hypoxia-Inducible Factor 1α with CREB-bindingprotein and/or p300, said method comprising: contacting the cell with anoxopiperazine according to claim 1 under conditions effective to reducetranscription of the gene.
 16. A method of treating in a subject adisorder mediated by interaction of Hypoxia-Inducible Factor 1α withCREB-binding protein and/or p300, said method comprising: administeringto the subject an oxopiperazine according to claim 1 under conditionseffective to treat the disorder.
 17. A method of reducing angiogenesisin a tissue, said method comprising: contacting the tissue with anoxopiperazine according to claim 1 under conditions effective to reduceangiogenesis in the tissue.
 18. A method of inducing apoptosis of acell, said method comprising: contacting the cell with an oxopiperazineaccording to claim 1 under conditions effective to induce apoptosis ofthe cell.
 19. A method of decreasing survival and/or proliferation of acell, said method comprising: contacting the cell with an oxopiperazineaccording to claim 1 under conditions effective to decrease survivaland/or proliferation of the cell.
 20. An oxopiperazine of Formula IC:

wherein: R₁ is hydrophobic and is an amino acid side chain, OR, SR, analkyl, or an aryl; wherein R is independently H, an alkyl, or an aryl;R₂ is an amino acid side chain, OR, SR, an alkyl, or an aryl; wherein Ris independently H, an alkyl, or an aryl; wherein R₂ is either a leucineside chain or R₂ is longer than a leucine side chain by at least onebackbone methylene; and with the proviso that R₁ and R₂ are not both anaryl; R₃ is hydrophobic and is an amino acid side chain, SR, or analkyl; wherein R is independently H, an alkyl, or an aryl; R₄ is ahydrogen bond donor or an amide; R₅ is a hydrophobic amino acid sidechain; each R₆ is independently H, N(R)₂, OR, halogen, an alkyl, or anaryl; wherein each R is independently H, an alkyl, or an aryl; R₇ is asolubilizing group, a hydrophobic amino acid side chain, H, N(R)₂, OR,SR, halogen, an alkyl, or an aryl; wherein each R is independently H, analkyl, or an aryl; X₁ is H, N(R)₂, OR, COR′, CO₂R′, CONR′, an alkyl, anaryl, an arylalkyl, a cycloalkyl, a heteroaryl, a peptide of 1 to about10 amino acid residues, a protecting group for protection of an amine, asolubilizing group, a targeting moiety, or a tag; wherein each R isindependently H, an alkyl, or an aryl; and wherein R′ is H, an alkyl, anaryl, an arylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or atag; Z is N; each A₁-W₁ is:

and Y is OR′, N(R′″)₂, an alkyl, an aryl, an arylalkyl, a cycloalkyl, aheteroaryl, an amino acid, a peptide of 1 to about 10 amino acidresidues, a protecting group for protection of a carboxylic acid, atargeting moiety, or a tag; wherein R′ is H, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag; andwherein each R′″ is independently H, CO₂R′, CONR′, an alkyl, an aryl, anarylalkyl, a cycloalkyl, a heteroaryl, a targeting moiety, or a tag. 21.The oxopiperazine according to claim 20, wherein R₁ is OR, SR, a C₃-C₆alkyl, an aryl, or a side chain of an amino acid selected from the groupconsisting of leucine, methionine, and homoleucine; R₂ is OR, SR, aC₃-C₆ alkyl, an aryl, or a side chain of an amino acid selected from thegroup consisting of norleucine, methionine, leucine, and homoleucine; R₄is a side chain of an amino acid selected from the group consisting ofglutamine, asparagine, and homoserine; R₅ is a side chain of an aminoacid selected from the group consisting of alanine, valine, and serine;and Y is OH, OR′, NEM′, NR′2, or NH₂.
 22. A pharmaceutical formulationcomprising: an oxopiperazine according to claim 20 and apharmaceutically acceptable vehicle.
 23. The pharmaceutical formulationaccording to claim 22, wherein R₁ is OR, SR, a C₃-C₆ alkyl, an aryl, ora side chain of an amino acid selected from the group consisting ofleucine, methionine, and homoleucine; R₂ is OR, SR, a C₃-C₆ alkyl, anaryl, or a side chain of an amino acid selected from the groupconsisting of norleucine, methionine, leucine, and homoleucine; R₄ is aside chain of an amino acid selected from the group consisting ofglutamine, asparagine, and homoserine; R₅ is a side chain of an aminoacid selected from the group consisting of alanine, valine, and serine;and Y is OH, OR′, NEM′, NR′₂, or NH₂.